http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Ebersaba&year=&month=2010.igem.org - User contributions [en]2024-03-29T05:22:04ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T21:20:43Z<p>Ebersaba: /* SEPTEMBER */</p>
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
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----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR 1 & Pst 1 Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
<p>[[Image:GFP from iGEM (E0040) paint.jpg|500px]]</p><br />
* The "GFP E0040" gel analysis was successful for the expected bands of GFP showed at ~720 bp when digested with EcoR 1 & Pst 1 (labeled Cut #1, Cut #2, Cut#3, Cut#4).<br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
<p>[[Image:GFP PCR product.jpg|500px]]</p><br />
* The "GFP PCR" gel analysis was successful for the expected bands of GFP + Primers showed at ~ 804bp for all PCR products (labeled 1 ng, 5ng, 10ng, & ~826ng)<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p><br />
* The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp. <br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony #17 (Digested/Cut sample labeled C17) the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony #17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested rd29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** rd29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [rd29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [rd29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [rd29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
https://static.igem.org/mediawiki/2010/b/bf/CcdB%2BpSB1C3_gel.png<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/File:Elaine2.jpgFile:Elaine2.jpg2010-10-27T21:09:14Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:Nevada/fundraising_sponsorshipsTeam:Nevada/fundraising sponsorships2010-10-27T20:58:24Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Funraising.png|border|left|900px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<br />
== Fundraising and Sponsorships ==<br />
<br />
<html><img src="" class="shadow" style="float:right"></html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg"><img src="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg" class="shadow" style="float:left;width:300px;margin:10px"></a></html><p>In order for the Nevada iGEM team to complete the necessary Bio Bricks for competition, a large source of financial capital was needed. It is a result of the generosity of Nevada INBRE that a majority of the tools and equipment (including, but not limited to, oligonucleotides, plasmids, cell lines, enzymes, and buffers) were met in a timely manner. In addition, INBRE covered the necessary expenses of travel, lodgings, and registration costs for a team of 10 individuals to the Jamboree in Boston, MA. All in all, INBRE provided over $16,000 in funding and without them this project would not have been possible.<br />
<br><br />
<br>The Department of Biochemistry and Molecular Biology was also exceptionally supportive in providing equipment, supplies, lab space and in attending our fundraising events. As the Nevada team approaches the Jamboree, the department has generously offered to cover any unexpected costs that may arise. The Nevada team thanks them for their ongoing support of this project. We also want to thank Invitrogen and Promega for their teaching discounts on necessary supplies. In addition, we want to thank Diana Long at Promega for sending us free enzyme when we were desperately short on funding. <br />
<br> <br />
<br><br />
<br>Finally, the iGEM team turned to the Reno community for help and support. Through cooperative efforts and local generosity, the Nevada team hosted a bar crawl, an ice cream social, notebook sales and a theatrical play in the Reno community. Through the selection of strategic locations and dates during summer and fall, these fundraising efforts helped attract business to the local economy and spark the interest of young scientists in and around the university community. The feedback and support was overwhelmingly positive. The events were successful in not only raising money, but also raising awareness about synthetic biology and the University of Nevada’s growing biotechnology research. Thanks to the community’s efforts, the Nevada team was able to raise an additional $1,500. In a state devastated by high unemployment and low revenue from tourism, the support and generosity that was received was incredible. It is thanks to the efforts of local entrepreneurs in the community’s entertainment district and university students that allowed the team to raise the necessary money to help alleviate the costs of doing science.<br />
</p><br />
<br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org/ Sparks Seventh-day Adventist Church]</span> for the $1,000 donation in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T20:57:48Z<p>Ebersaba: </p>
<hr />
<div>{{nevadamain}}<br />
== Abstract ==<br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
</html><br />
<br />
<br><br />
<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, the''' 2010 Nevada iGEM Team''' has three goals for this year’s competition. First, we are going to test the validity of utilizing Nicotiana tabacum (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. These cells can therefore be utilized as a quick proof-of-concept test model before moving synthetic constructs into plants of interest. We also aim to produce an iGEM-compatible plant-specific plasmid, several stress-inducible plant promoters, reporter genes containing Kozak sequences (ribosome binding sites) and terminators that conform to BioBrick standards. Lastly, we hope to measure the induction of these stress promoters in real-time by performing a fluorometry assay in which stress will be applied to NT cells and fluorescent output by a reporter (GFP) will be measured to detail induction in real time. This method has a distinct advantage over microarrays since microarrays are only one ‘snapshot’ in time.</p><br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org/ Sparks Seventh-day Adventist Church]</span> for the $1,000 donation in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/CD2InducibleTeam:Nevada/CD2Inducible2010-10-27T19:37:05Z<p>Ebersaba: </p>
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<div>{{Nevada_css}}<br />
[[Image:Picture 13.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
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<p>&nbsp;</p><br />
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== Promoters ==<br />
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<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB 1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">rd29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png '''Cadmium Inducible Promoter''' [[Team:Nevada/registry submissions]]<br />
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<html><a href="https://static.igem.org/mediawiki/2010/8/8f/Hileary.jpg"><img src="https://static.igem.org/mediawiki/2010/8/8f/Hileary.jpg" class="shadow" style="float:left;width:200px;margin:10px"></a><br />
</html><br />
<p>Heavy metal contamination is an important environmental issue. Heavy metals can contaminate soil and water sources in areas where mining and various industrial proccesses have occurred. These metals can then be absorbed into plants which are subsequently eaten by various animals. Heavy metals are usually not excreted readily and are retained within the body of an animal that has consumed a contaminated food source. Cadmium in particular is not excreted readily from the mammals and is known to cause various etiologies stemming from its build-up in organs (Gobe and Cramer). In order to develop a mock cadmium-sensing system in plants the promoter for the Cd-transporter gene AtMRP3 (At3g13080) from A. thaliana was transformed into N. tabacum cells. AtMRP3 is utilized by the plant to sequester Cd2+ in the vacuole, which is thought to prevent the cation from interfering with various biological processes (Bovet et al.). Besides being highly induced by cadmium, AtMRP3 has also shown similar induction patterns when plants were subjected to arsenic or lead, thusly making it a useful sensor for various heavy metal soil contaminants.</p><br />
<p>&nbsp;</p><br />
<p>AtMRP3 will be the first plant-compatible heavy metal promoter available to the iGEM registry. This promoter could be coupled with a myriad of reporters to indicate whether or not plants are experiencing any type of stress due to the presence of cadmium or other heavy metals.</p><br />
<br><br />
'''References'''<br />
<br>'''Bovet et al.''' Transcript levels of AtMRP3 after cadmium treatment: induction of AtMRP3. Plant, Cell and Environment., 26: 371-381, 2003.<br />
<br>'''Gobe and Cramer.''' Mitochondria, reactive oxygen species and cadmium toxicity in the kidney. Toxicology Letters., 198: 49-55, 2010.<br />
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<!--- The Mission, Experiments ---><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/35STeam:Nevada/35S2010-10-27T19:36:49Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada35S}}<br />
<br />
<br />
== Promoters ==<br />
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<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">rd29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
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https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''35S Promoter''' [[Team:Nevada/registry submissions]]<br />
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<br />
<html><a href="https://static.igem.org/mediawiki/2010/2/2f/Picture_16.png"><img src="https://static.igem.org/mediawiki/2010/2/2f/Picture_16.png" style="float:left;width:200px;margin:10px"></a></html><p>The 35S promoter is frequently used as a constitutive promoter in plant research, primarily Arabidopsis experiments, and has been demonstrated to work in Nicotiana tabacum (Kuluev et al, 2010). This promoter normally drives transcription of the Cauliflower mosaic virus genome and shows no tissue or developmental specificity (Keller et al, 2002). For these reasons, the 2010 Nevada iGEM team modified the 35S promoter to conform to BioBrick standards, providing a reliable constitutive promoter to future iGEM teams wishing to engineer plants.<br />
<br><br />
<br>'''References'''<br />
<br>'''Keller, M., Haas, M., Bureau, M., Geldreich, A. and Yot, P.''' (2002) Cauliflower mosaic virus: still in the news.<br />
Molecular Plant Pathology, 3(6), 419–429.<br />
<br>'''Kuluev, B. R., Knyazev, A. V., Lebedev, P. Ya., Iljassowa, A. A. and Chemeris, A. V.''' (2010) Construction of Hybrid Promoters of Caulimoviruses and Analysis of Their Activity in Transgenic Plants. Russian Journal of Plant Physiology, Vol. 57, No. 4, 582-589.</p><br />
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<!--- The Mission, Experiments ---><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/RD29ATeam:Nevada/RD29A2010-10-27T19:36:31Z<p>Ebersaba: </p>
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<div>{{nevadaRD29A}}<br />
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== Promoters ==<br />
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<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">rd29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''RD29A Promoter + Strong Plant Kozak (RBS) + RFP''' [[Team:Nevada/registry submissions]]<br />
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<html><a href="https://static.igem.org/mediawiki/2010/c/c1/Picture_17.png"><img src="https://static.igem.org/mediawiki/2010/c/c1/Picture_17.png" class="shadow" style="float:left;width:200px;margin:10px"></a><br />
</html><br />
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<p>In our project, we designed the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter and the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP. These both came from the synthetic design that contained the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP in the pMA vector.<br />
The <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> promoter was isolated by the use of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> designed primers in PCR. This was blunt end Topo cloned, digested with EcoR 1 and Pst 1 sites, and was then ligated to the pSB1C3 vector.<br />
The design of the <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter">rd29A</a></html> Promoter + Strong Plant Kozak (RBS) + RFP involved the ligation to the pSB1C3 vector using the EcoR 1 and Pst 1 sites. This composite part contains both the promoter and the reporter gene. This functions when the promoter is activated during environmental stress, which would then allow the expression of red fluorescence in plants that can be used as a warning signal to farmers that their plants are under stress.</p><br />
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<br><br />
<br>'''References'''<br />
<br><br />
'''Cong L, Zheng H, Zhang Y, Chai T.''' Arabidopsis DREB1A confers high salinity tolerance and regulates the expression of GA dioxygenases in Tobacco. Plant Science [serial online]. February 2008;174(2):156-164. Available from: Academic Search Premier, Ipswich, MA. Accessed October 24, 2010.<br />
<br><br />
'''Babak B, Akira K, Fevziye C, Mie K, Kazuko Y, Kazuo W.''' Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato. Plant Cell Reports [serial online]. August 26, 2007;26(8):1275-1282. Available from: Academic Search Premier, Ipswich, MA. Accessed October 25, 2010.<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/DREB1CTeam:Nevada/DREB1C2010-10-27T19:36:13Z<p>Ebersaba: </p>
<hr />
<div>{{nevadaDREB1C}}<br />
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== Promoters ==<br />
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<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">rd29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png '''DREB1C promoter''' [[Team:Nevada/registry submissions]] <br />
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<p><html><a href="https://static.igem.org/mediawiki/igem.org/d/d1/Chris_Igem.jpg"><img src="https://static.igem.org/mediawiki/igem.org/d/d1/Chris_Igem.jpg" class="shadow" style="float:left;width:200px;margin:10px"></a><br />
</html> The <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> iGEM promoter is derived from a transcription factor that is up-regulated by cold stress and down-regulated by circadian controls to prevent plant growth retardation due to the buildup of <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> (Dehydration Response Element Binding Protein) during the day (the cause of dwarfism). The promoter region for <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> begins just upstream of the translational start sequence (ATG) and contains 463 bp of the upstream sequence with six ADA independent, <i>cis</i>-acting elements for the up-regulation during cold stress and circadian controlled down-regulation.</p><br />
<br />
<p>The <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> iGEM promoter was isolated using custom primers directly from the <i>Arabidopsis thaliana</i> genome via PCR, blunt end Topo cloned, and then ligated to pSB1C3 using EcoR1 and Pst1 sites.</p><br />
<br />
<br>'''References'''<br><br />
'''Kazuo Nakashima and Kazuko Yamaguchi-Shinozakia''', Regulons involved in osmotic stress-responsive and cold stress-responsive gene expression in plants, Physiologia Plantarum 126: 62–71. 2006<br />
'''Satoshi Kidokoro, Kyonoshin Maruyama, Kazuo Nakashima, Yoshiyuki Imura2, Yoshihiro Narusaka3, Zabta K. Shinwari4, Yuriko Osakabe, Yasunari Fujita, Junya Mizoi, Kazuo Shinozaki, and Kazuko Yamaguchi-Shinozaki''', The Phytochrome-Interacting Factor PIF7 Negatively Regulates DREB1 Expression under Circadian Control in Arabidopsis, Plant Physiol. Vol. 151, 2009<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T19:34:11Z<p>Ebersaba: </p>
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<div>{{nevadamain}}<br />
== Abstract ==<br />
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<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
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<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, the''' 2010 Nevada iGEM Team''' has three goals for this year’s competition. First, we are going to test the validity of utilizing Nicotiana tabacum (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. These cells can therefore be utilized as a quick proof-of-concept test model before moving synthetic constructs into plants of interest. We also aim to produce an iGEM-compatible plant-specific plasmid, several stress-inducible plant promoters, reporter genes containing Kozak sequences (ribosome binding sites) and terminators that conform to BioBrick standards. Lastly, we hope to measure the induction of these stress promoters in real-time by performing a fluorometry assay in which stress will be applied to NT cells and fluorescent output by a reporter (GFP) will be measured to detail induction in real time. This method has a distinct advantage over microarrays since microarrays are only one ‘snapshot’ in time.</p><br />
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<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
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<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
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<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org/ Sparks Seventh-day Adventist Church]</span> who donated $1,000 in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/fundraising_sponsorshipsTeam:Nevada/fundraising sponsorships2010-10-27T19:33:03Z<p>Ebersaba: /* Fundraising and Sponsorships */</p>
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== Fundraising and Sponsorships ==<br />
<br />
<html><img src="" class="shadow" style="float:right"></html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg"><img src="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg" class="shadow" style="float:left;width:300px;margin:10px"></a></html><p>In order for the Nevada iGEM team to complete the necessary Bio Bricks for competition, a large source of financial capital was needed. It is a result of the generosity of Nevada INBRE that a majority of the tools and equipment (including, but not limited to, oligonucleotides, plasmids, cell lines, enzymes, and buffers) were met in a timely manner. In addition, INBRE covered the necessary expenses of travel, lodgings, and registration costs for a team of 10 individuals to the Jamboree in Boston, MA. All in all, INBRE provided over $16,000 in funding and without them this project would not have been possible.<br />
<br><br />
<br>The Department of Biochemistry and Molecular Biology was also exceptionally supportive in providing equipment, supplies, lab space and in attending our fundraising events. As the Nevada team approaches the Jamboree, the department has generously offered to cover any unexpected costs that may arise. The Nevada team thanks them for their ongoing support of this project. We also want to thank Invitrogen and Promega for their teaching discounts on necessary supplies. In addition, we want to thank Diana Long at Promega for sending us free enzyme when we were desperately short on funding. <br />
<br> <br />
<br><br />
<br>Finally, the iGEM team turned to the Reno community for help and support. Through cooperative efforts and local generosity, the Nevada team hosted a bar crawl, an ice cream social, notebook sales and a theatrical play in the Reno community. Through the selection of strategic locations and dates during summer and fall, these fundraising efforts helped attract business to the local economy and spark the interest of young scientists in and around the university community. The feedback and support was overwhelmingly positive. The events were successful in not only raising money, but also raising awareness about synthetic biology and the University of Nevada’s growing biotechnology research. Thanks to the community’s efforts, the Nevada team was able to raise an additional $1,500. In a state devastated by high unemployment and low revenue from tourism, the support and generosity that was received was incredible. It is thanks to the efforts of local entrepreneurs in the community’s entertainment district and university students that allowed the team to raise the necessary money to help alleviate the costs of doing science.<br />
</p><br />
<br />
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<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org/ Sparks Seventh-day Adventist Church]</span> who donated $1,000 in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/fundraising_sponsorshipsTeam:Nevada/fundraising sponsorships2010-10-27T19:31:19Z<p>Ebersaba: /* Fundraising and Sponsorships */</p>
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{{Nevada_topbar}}<br />
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== Fundraising and Sponsorships ==<br />
<br />
<html><img src="" class="shadow" style="float:right"></html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg"><img src="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg" class="shadow" style="float:left;width:300px;margin:10px"></a></html><p>In order for the Nevada iGEM team to complete the necessary Bio Bricks for competition, a large source of financial capital was needed. It is a result of the generosity of Nevada INBRE that a majority of the tools and equipment (including, but not limited to, oligonucleotides, plasmids, cell lines, enzymes, and buffers) were met in a timely manner. In addition, INBRE covered the necessary expenses of travel, lodgings, and registration costs for a team of 10 individuals to the Jamboree in Boston, MA. All in all, INBRE provided over $16,000 in funding and without them this project would not have been possible.<br />
<br><br />
<br>The Department of Biochemistry and Molecular Biology was also exceptionally supportive in providing equipment, supplies, lab space and in attending our fundraising events. As the Nevada team approaches the Jamboree, the department has generously offered to cover any unexpected costs that may arise. The Nevada team thanks them for their ongoing support of this project. We also want to thank Invitrogen and Promega for their teaching discounts on necessary supplies. In addition, we want to thank Diana Long at Promega for sending us free enzyme when we were desperately short on funding. <br />
<br> <br />
<br><br />
<br>Finally, the iGEM team turned to the Reno community for help and support. Through cooperative efforts and local generosity, the Nevada team hosted a bar crawl, an ice cream social, notebook sales and a theatrical play in the Reno community. Through the selection of strategic locations and dates during summer and fall, these fundraising efforts helped attract business to the local economy and spark the interest of young scientists in and around the university community. The feedback and support was overwhelmingly positive. The events were successful in not only raising money, but also raising awareness about synthetic biology and the University of Nevada’s growing biotechnology research. Thanks to the community’s efforts, the Nevada team was able to raise an additional $1,500. In a state devastated by high unemployment and low revenue from tourism, the support and generosity that was received was incredible. It is thanks to the efforts of local entrepreneurs in the community’s entertainment district and university students that allowed the team to raise the necessary money to help alleviate the costs of doing science.<br />
</p><br />
<br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
Thank you to the <span style="font-weight:bold;">[http://sparks22.adventistchurchconnect.org//Default.aspx Sparks Seventh-day Adventist Church]</span> who donated $1,000 in response to the sponsorship letters.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/Team_Nevada:_Plant_SummitTeam:Nevada/Team Nevada: Plant Summit2010-10-27T19:26:37Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Plant Summit UNR.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== 2010 iGEM Plant Summit ==<br />
<br><br />
<p>On '''Sunday November 7th''', the iGEM Nevada Team is organizing the first '''iGEM Plant Summit'''. The goal of this summit is to develop a network of iGEM investigators working on plant systems that can help promote the adoption of plants by the larger iGEM community. We will discuss the initiation of a coordinated effort to develop iGEM compatible resources (i.e. plant promoters, reporter genes, and transformation vectors) and standardized plant transformation, maintenance and safety protocols. The pros and cons of various plant systems will be discussed. Furthermore, because, genetically engineered plants require more time to create than genetically engineered bacteria or fungi, we will also discuss strategies that will allow for the completion of plant related projects within the time constraint of the iGEM competition. <br />
</p><br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/fundraising_sponsorshipsTeam:Nevada/fundraising sponsorships2010-10-27T19:26:07Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Funraising.png|border|left|900px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<br />
== Fundraising and Sponsorships ==<br />
<br />
<html><img src="" class="shadow" style="float:right"></html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg"><img src="https://static.igem.org/mediawiki/2010/9/9b/12859_544382805569_23800361_31674393_4924904_n.jpg" class="shadow" style="float:left;width:300px;margin:10px"></a></html><p>In order for the Nevada iGEM team to complete the necessary Bio Bricks for competition, a large source of financial capital was needed. It is a result of the generosity of Nevada INBRE that a majority of the tools and equipment (including, but not limited to, oligonucleotides, plasmids, cell lines, enzymes, and buffers) were met in a timely manner. In addition, INBRE covered the necessary expenses of travel, lodgings, and registration costs for a team of 10 individuals to the Jamboree in Boston, MA. All in all, INBRE provided over $16,000 in funding and without them this project would not have been possible.<br />
<br><br />
<br>The Department of Biochemistry and Molecular Biology was also exceptionally supportive in providing equipment, supplies, lab space and in attending our fundraising events. As the Nevada team approaches the Jamboree, the department has generously offered to cover any unexpected costs that may arise. The Nevada team thanks them for their ongoing support of this project. We also want to thank Invitrogen and Promega for their teaching discounts on necessary supplies. In addition, we want to thank Diana Long at Promega for sending us free enzyme when we were desperately short on funding. <br />
<br> <br />
<br><br />
<br>Finally, the iGEM team turned to the Reno community for help and support. Through cooperative efforts and local generosity, the Nevada team hosted a bar crawl, an ice cream social, notebook sales and a theatrical play in the Reno community. Through the selection of strategic locations and dates during summer and fall, these fundraising efforts helped attract business to the local economy and spark the interest of young scientists in and around the university community. The feedback and support was overwhelmingly positive. The events were successful in not only raising money, but also raising awareness about synthetic biology and the University of Nevada’s growing biotechnology research. Thanks to the community’s efforts, the Nevada team was able to raise an additional $1,500. In a state devastated by high unemployment and low revenue from tourism, the support and generosity that was received was incredible. It is thanks to the efforts of local entrepreneurs in the community’s entertainment district and university students that allowed the team to raise the necessary money to help alleviate the costs of doing science.<br />
</p><br />
<br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T19:25:38Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR 1 & Pst 1 Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
<p>[[Image:GFP from iGEM (E0040) paint.jpg|500px]]</p><br />
* The "GFP E0040" gel analysis was successful for the expected bands of GFP showed at ~720 bp when digested with EcoR 1 & Pst 1 (labeled Cut #1, Cut #2, Cut#3, Cut#4).<br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
<p>[[Image:GFP PCR product.jpg|500px]]</p><br />
* The "GFP PCR" gel analysis was successful for the expected bands of GFP + Primers showed at ~ 804bp for all PCR products (labeled 1 ng, 5ng, 10ng, & ~826ng)<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p><br />
* The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp. <br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony #17 (Digested/Cut sample labeled C17) the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony #17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
https://static.igem.org/mediawiki/2010/b/bf/CcdB%2BpSB1C3_gel.png<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/SafetyTeam:Nevada/Safety2010-10-27T19:25:11Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Safety.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
== Safety ==<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/c/cd/IMG_6042.JPG"><img src="https://static.igem.org/mediawiki/2010/c/cd/IMG_6042.JPG" class="shadow" style="float:left;width:200px;margin:10px"></a></html><br />
<br />
<p>'''Would any of your project ideas raise safety issues in terms of:'''</p><br />
<br />
<p>'''''Researcher Safety,'''''</p><br />
<br />
<p>Nicotiana tabacum - No. NT cells are common cell lines used for decades in plant research. No known hazards have been associated with NT cell research.<br />
<br />
Reporter genes – No. Fluorescent proteins are a staple of molecular biology, and no known hazards have been associated with their use. Our promoters also pose no threat.</p><br />
<br />
<br />
<p>'''''Public Safety,'''''</p><br />
<br />
<p>Nicotiana tabacum - No. We do not intend on developing the project in any way such that the public would encounter our project. Even so, in a hypothetical commercial development, NT cells are not expected to put the public at risk. NT cells are not viable outside a nutrient-rich environment.</p><br />
<br />
<p>None of the parts we intend to make, promoters or fluorescent reporters, have shown any health risks to date.</p><br />
<br />
<br />
<p>'''''Environmental Safety'''''</p><br />
<br />
<p>- No. While other plant models could conceivably cross with wild-type plants and generate unforeseeable hybrids, NT cells mitigate that risk. Because NT cells are incapable of sexual reproduction and can only proliferate through clonal propagation in their nutrient-rich media. Containment of the cells is easier to manage with less risk should the NT cells ever breach containment. Therefore, we would not expect any of our promoters or reporter genes to reach the environment. We are ensuring proper containment of transformed Agrobacterium.</p><br />
<br />
<br />
<p>'''Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''</p> <br />
<br />
<p>- No. Our reporter genes are standard fluorescent proteins used repeatedly not just in iGEM but research abroad. Our modified plasmid poses no risk to the researchers, public, or environment. The inducible promoters selected are found naturally in plants and are predicted to pose no risk.</p><br />
<br />
<br />
<p>'''Is there a local biosafety group, committee, or review board at your institution? <br />
<br />
If yes, what does your local biosafety group think about your project?'''</p><br />
<br />
<p>- Yes. The University of Nevada, Reno Institutional Biosafety Committee supports our project, especially with regard to the fact every member of the iGEM team completed training on NIH Guidelines on Recombinant Organisms. An additional safety course conducted by the University of Nevada, Reno Environmental Health & Safety Department trained each member in the certified Lab Safety Workshop on chemical safety, biological safety, chemical hygiene, ventilation, and waste management. Therefore, all iGEM members have been trained in the handling and disposal of transgenic bacteria.</p> <br />
<br />
<br />
'''Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
<br />
<p>The steps previously and currently taken by iGEM teams to design systems where modified organisms can be screened or terminated under certain conditions are excellent ways of providing safety. Certainly, controlling where and how organisms can grow helps to ensure modified organisms do not unintentionally interact with the environment. These are all internal controls, and while 99.9% reliable, we believe the risk of unforeseen mutation cannot be overlooked. Several systems of control will have a few layers of regulation, but there could likely be one nexus of vulnerability that the whole system hinges upon, and the question must be asked what happens if that node of control fails? A dysfunctional fluorescent protein may not deserve attention, but genes regulating proliferation, reproduction, or repressors could cause bacteria, fungi, or plants to escape containment. Adding layers of regulation do help, but ultimately they affect the quality of the plasmids or genes used and may ultimately hinder the goals set for the project.</p><br />
<br />
<p>Therefore, we propose one additional way of providing safety in an iGEM doomsday scenario to help in the event of some future iGEM bacteria that infects humans or plants, affecting public health or crops. Perhaps, as iGEM grows and commercial applications become apparent, for plant projects, iGEM could require a constitutively expressed fluorescent protein alongside any novel gene to track the plants. iGEM-compatible internal controls have been designed, for example Harvard’s project, but in the rare event those mechanisms fail, a constitutively expressed fluorescent protein would be an easily identifiable way of tracking iGEM plants.</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/ModelingTeam:Nevada/Modeling2010-10-27T19:24:54Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Modeling.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<br />
== Modeling ==<br />
https://static.igem.org/mediawiki/2010/a/aa/Pending.png<br />
----<br />
=== Introduction ===<br />
<br />
<p>Plant stress responses are often cascades involving hundreds of genes and gene products. The possible interactions in these cascades are astronomical. Therefore, the 2010 Nevada iGEM team worked with Bioinformatics Professor, Karen Schlauch, to use a computational method that could quickly analyze possible transcriptional regulation pathways, using either microarray data or data from continuous fluorometry experiments. In conjunction with the use of tobacco BY-2 (NT1) cells, this method could allow for even greater time efficiency in identifying important aspects of gene networks in plants. The method was intended to allow for easier identification of promoters useful to the team’s objective of creating remote plant biosensors. The method uses a Boolean network approach to examine the gene network and its possible regulatory system. We first viewed transcripts as “on” when above a threshold value and “off” for lesser values. All Boolean networks that could generate our dataset were generated and evaluated. In this manner, we were able to look at all possible interactions between genes based on the Boolean approach.</p><br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
=== The DREB1 Pathway ===<br />
<br><br />
<br><br />
<br><br />
<br />
=== Boolean Networks ===<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Data ===<br />
<br />
When it became clear that the team would not have sufficient time to perform fluorometry experiments and analyze data by November, it was decided that microarray experiments published to internet databases would have to do. All data was originally obtained from a 24-hour time course microarray experiment performed by Jian-Kang Zhu, ''et al,'' and published on the Gene Expression Omnibus database (Zhu, ''et al.'' 2005). This allowed for a proof-of-concept to see if the Boolean network would support what was known about the DREB1 pathway from published literature. <br />
<br />
[https://2010.igem.org/Team:Nevada/Original_Data<u>Click here to see the initial data set</u>]<br />
<br />
Because this data consisted of only four time points, all eight genes had similar Boolean values at each time point. Therefore, the Boolean functions for each were essentially the same and numbered in the billions. This provided little data for interpretation. Several methods were used to tease out differences in expression, so that the time courses would be sufficiently different. First, the threshold value for "on" was raised to 2^3.5 rather than 4. Second, The data was interpolated to estimate extra time points within the 24-hour time course. Finally, the number of inputs for each gene was limited to four, as it was deemed unlikely that any gene in this network was receiving input from 5 or more.<br />
<br />
[https://2010.igem.org/Team:Nevada/Interpolated_Data<u>Click here to see the interpolated data set and associated Boolean functions</u>]<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
=== Results ===<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png"><img src="https://static.igem.org/mediawiki/2010/d/d8/IGEM_DREB1_pathway.png" style="float:left;width:200px;margin:10px"></a></html><br />
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<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=== References ===<br />
<br />
Zhu, J-K., Lee, B., Henderson, D. (2005) The Arabidopsis Cold-Responsive Transcriptome and Its Regulation by ICE1. ''Plant Cell.'' Vol. 17, Issue 11, p3155-3175.<br />
<br />
<br />
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<br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/Transgenic_PlantsTeam:Nevada/Transgenic Plants2010-10-27T19:24:30Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Transgenic Plants.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== Transgenic Plants: into the Wild ==<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants: into the Wild</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<p>'''Technological Advances from Genetically Engineered Plants'''<br />
<br><br />
Since the initial development of Agrobacterium transformation systems, many plant species including tobacco, tomato, potato, rice, soybean, mint, melon, cucumber, pine and poplar trees, and many others have been transformed using this ingenious bacterial vector. Important traits have been engineered into plants including pest and weed resistance, increased nutritional value, environmental stress tolerance, the production of pharmaceutical and industrial proteins, and the production of bioactive secondary chemical compounds. Our ability to genetically engineer plants has revolutionized agriculture by increasing crop yields while drastically decreasing the application of herbicides and pesticides. This technology is necessary to allow farmers to produce sufficient food for a growing global population. Furthermore, plants are currently being engineered to produce fuel and chemical alternatives to petroleum based products. Because plants are net consumers of atmospheric carbon dioxide, they are currently being seen as a means to sequester greenhouse gases while at the same time replacing petroleum and coal as chemical feedstocks. <br />
<br><br />
<br>However, there has been recent controversy concerning the use of transgenic plants and organisms. These issues include economical, environmental, ethical, and health concerns. We have developed the following graphics outlining and discussing a short history and issues concerning GMOs. This images has been made available to help educate and for use by other participants.</p><br />
<br />
<html><a href=""><img src="" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/BY-2_(NT1)Transformation_ProtocolTeam:Nevada/BY-2 (NT1)Transformation Protocol2010-10-27T19:23:57Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:NT cell transformations.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== BY-2 (NT1) Cell Transformation Protocol ==<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants: into the Wild</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG"><img src="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG" class="shadow" style="float:left;width:500px;margin:10px"></a><br />
</html><br />
<br />
<br />
<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">NT Cell Protocol</span></p><br />
<br />
<p>'''CARE OF BY-2 (NT1) CELL CULTURES'''</p><br />
(adapted from a letter by Dr. Michael Sullivan) <br />
To readapt a culture on plates, simply transfer some of the cells back into liquid <br />
media. We usually pipette the cell suspension up and down to break up any clumps. It <br />
may be best to start out with a smaller culture volume when you first go back to liquid; <br />
BY-2 seems to be a bit happier if it isn't seeded too thinly. Allow the culture to grow <br />
until it is the consistency of thin applesauce. At this point, you should be able to go to <br />
a 50 ml culture and start subculturing as described below. In our experience, wild-type <br />
BY-2 readapts quickly to liquid to give a smooth culture; transformed lines tend to be more <br />
variable, with some producing smooth cultures, some producing clumpy ones, and some going <br />
back and forth between these two states. We've found that clumpy cultures do not interfere <br />
with our half-live measurements, although manipulating them can be a bit more difficult. <br />
<br />
We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C <br />
with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement, many <br />
people grow these cells in regular flasks with no problem. We subculture them once a week <br />
by transferring 5% of the culture to fresh media. We generally maintain two flasks <br />
(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) <br />
in case one of the cultures crashes. Also, you can maintain the culture on a plate. <br />
Note that, for liquid cultures of transformed lines, we usually use a smaller culture volume, <br />
e.g. 10 ml, for convenience. <br />
<br />
<br />
'''NT KC MEDIA - LIQUID OR PLATES<br />
NT LIQUID:''' <br />
(amounts are for 1L of media): <br />
750 ml H2O <br />
4.3 g MS salts (add slowly to liquid) <br />
30 g Sucrose <br />
50 ml 20X MES pH 5.7 <br />
10 ml B1 -inositol <br />
3 ml Miller's I <br />
10 ml 2,4-D, 10-4 M <br />
pH to 5.7 with 0.1 N KOH <br />
Bring volume to 1000ml <br />
Autoclave <br />
<br />
'''SOLID MEDIA:''' <br />
For plates only: Add to flasks 7 g/ L Phytagar before autoclaving <br />
Add kanamycin (100 mg/ml) <br />
Add carbenicillin (250 mg/ml) <br />
<br />
'''MEDIA COMPONENTS:''' <br />
Miller's I: 60 g KH2 PO4 per liter <br />
20 X MES: 10 g MES per liter, pH to 5.7 with 1N KOH <br />
B1 - Inositol: 0.1 g Thiamine, 10.0 g myo-inositol, H2O to final volume of 1 liter <br />
<br />
<br />
<br />
<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">Transformation Protocol</span></p><br />
<p>'''BY-2 (NT1) Cell Transformation with Agrobactrium'''</p><br />
<p>'''Day 1:''' </p><br />
<p>1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary. </p><br />
<br />
<p>'''Day 2:''' </p><br />
<p>2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. </p><br />
<br />
3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished. <br />
<br />
4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation. <br />
<br />
5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA. <br />
<br />
6. Wrap plates with parafilm and incubate for 3 days at 28 degrees C. <br />
<br />
<p>'''Day 5:''' </p><br />
7. To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml carbenicillin (NTC).<br />
<br />
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC. <br />
<br />
9. Centrifuge at 1K for 4 minutes at room temperature in a swinging bucket rotor. <br />
<br />
10. Aspirate off liquid using pipet capped with a sterile blue 1 ml top. Change tip for each sample.<br />
<br />
11. Resuspend in 50 ml NTC and repeat spin. <br />
<br />
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are fairly sensitive to carb require fewer washes. <br />
<br />
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml on 2 NTKC (kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no longer lots of fluid on the plates (leave the plates open in the hood a few minutes of necessary), wrap them in parafilm.<br />
<br />
14. Incubate at 28 degrees C. Transformants should be large enough to transfer to fresh NTKC plates in 3-4 weeks. <br />
<br />
'''Supplies for each transformation (Remember the controls):''' <br />
<br />
Day 1 Supplies: <br />
LB + appropriate drugs<br />
Agrobacterium containing plasmid for transformation <br />
<br />
Day 2 Supplies: <br />
1 ml Agrobacterium overnight culture <br />
4 ml BY-2 cells - 3 days post subculture <br />
4 ul acetosyringone (20 mM) found in the (-20) refrigerator freezer <br />
pipets and pipetman <br />
1 petri plate <br />
<br />
Day 5 Supplies: <br />
200 ml NTC liquid <br />
50 ml conical tube <br />
swinging bucket centrifuge at room temp aspiration setup with 5 ml pipet capped with 1 ml blue tip <br />
2 NTKC plates <br />
pipetmen and tips <br />
<br />
<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG"><img src="https://static.igem.org/mediawiki/2010/4/4f/IMG_6028.JPG" class="shadow" style="float:left;width:430px;margin:10px"></a><br />
</html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/3/3d/IMG_6035.JPG"><img src="https://static.igem.org/mediawiki/2010/3/3d/IMG_6035.JPG" class="shadow" style="float:right;width:430px;margin:10px"></a></html><br />
<br />
<br />
<br />
----<br />
<p>'''Left:''' NT Cell Culture after about 5 days. '''Right:''' NT Cell Transformation with RD29A (Cold, Drought, Salt) Inducible Promoter.</p><br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
<br />
<br />
----<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/ResultsTeam:Nevada/Results2010-10-27T19:23:18Z<p>Ebersaba: </p>
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<div>{{Nevada_css}}<br />
[[Image:Results UNR Final.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<br />
== NT Cell Transformation Results ==<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants: into the Wild</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG"><img src="https://static.igem.org/mediawiki/2010/a/a6/IMG_6032.JPG" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
</html><br />
'''Plated NT Cells Immediately after Transformation'''<br />
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<html><a href="https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells(2).jpg"><img src="https://static.igem.org/mediawiki/2010/6/60/Igem_ntcells(2).jpg" class="shadow" style="float:center;width:400px;margin:10px"></a><br />
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'''Plated Transformed NT Cells after about 3 weeks of Cell Growth'''<br />
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<br />
<html><a href="https://static.igem.org/mediawiki/2010/8/89/NT_Results_UNR.png"><img src="https://static.igem.org/mediawiki/2010/8/89/NT_Results_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p>'''RD29A + RFP Transformed NT Cells after about 3 weeks of growth:''' The NT cells shown above were transformed with RD29A+RFP, a composite part in which the RD29A promoter is turned on due to external stimuli such as cold, drought or salinity. When the RD29A promoter is turned "on" it then turns on its reporter, in this case RFP. After about 3 weeks of NT cell growth, the transformed cells were "cold stressed" for about 3hrs in 4 degrees Celsius. The results shown indicate that when cold stressed, the transformed NT cells become fluorescent with RFP. Over the next few weeks we hope to see this signal grow stronger and test these NT cells' responsiveness to other stresses that induce the RD29A promoter.</p><br />
<br><br />
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<br><br />
<p>'''35S + GFP Transformed NT Cells:''' Currently, our transformed NT cells that contain the composite part, 35S + GFP, have not grow enough to determine whether or not the transformation was successful. The 35S promoter was designed as a constitutive control and would always be turned "on", thus maintaining a constant GFP fluorescent signal. Over the next few weeks we hope to see more cell growth and eventually transformed cells that produce green fluorescent protein constitutively.</p><br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/registry_submissionsTeam:Nevada/registry submissions2010-10-27T19:23:01Z<p>Ebersaba: </p>
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<div>{{Nevada_css}}<br />
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<p>&nbsp;</p><br />
== Registry Submissions ==<br />
<br />
<br />
<br />
<br />
<groupparts>iGEM010 Nevada</groupparts><br />
<br />
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<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
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!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/ccdBTeam:Nevada/ccdB2010-10-27T19:22:36Z<p>Ebersaba: </p>
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<p>&nbsp;</p><br />
<br />
== ccdB ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
'''ccdB minimal gene''' [[Team:Nevada/registry submissions]]<br />
<br />
----<br />
<html><a href="https://static.igem.org/mediawiki/2010/a/a0/Randy_and_Vadim.jpg"><img src="https://static.igem.org/mediawiki/2010/a/a0/Randy_and_Vadim.jpg" class="shadow" style="float:left;width:400px;margin:10px"></a></html><p>The ccdB gene is a known topoisomerase II poison and will kill most commercially available E. coli cell lines. The ccdB gene can be used as a selectable marker by placing it in the multicloning region of a plasmid and propagating it in ccdB resistant cell lines (e.g. DB3.1 from New England Biolabs). During cloning, the ccdB gene can be switched out for your gene of interest and transformed into a cell line that is susceptible to the toxic effects of ccdB (e.g NEBβ cells from New England Biolabs). Colonies that grow on plates should contain the plasmid and your gene of interest. If the ccdB gene is still present in the plasmid, the plasmid will kill any colonies where it is still present. This is an improvement to the current ccdB cell death gene part (BBa_P1010) as it does not contain the inactive ccdA gene or an unknown stuffer region.</p><br />
<p>&nbsp;</p><br />
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<p>'''Our Method:''' ccdB was amplified using Polymerase Chain Reaction (PCR) with primers designed to contain the specific BioBrick prefix and suffix. The amplified fragment was then TOPO-cloned into a PCR-Blunt II vector (Invitrogen). The vector was then cut with EcoRI and PstI restriction enzymes and the ccdB fragment was transformed into the iGEM compatible vector pSB1C3.<html><a href="https://static.igem.org/mediawiki/2010/0/05/CcdB_method.png"><img src="https://static.igem.org/mediawiki/2010/0/05/CcdB_method.png" class="shadow" style="float:center;width:850px;margin:10px"></a></html></p><br />
<p>&nbsp;</p><br />
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<br />
<p>'''Our Results:'''<br />
<br><br />
*ccdB was transformed in three different cell lines: NEB10β cells (New England Biolabs), Omni Max 2 cells (Invitrogen), and DB3.1 cells (New England Biolabs). NEB10β and Omni Max 2 cell lines are not resistant to the toxic properties of ccdB. No colonies were present in those two cell lines but were present in DB3.1 cell lines (Left). <br />
<br><br />
*PCR gel confirming the presence of ccdB in amplified sample(Right). <html><a href="https://static.igem.org/mediawiki/2010/6/63/Ccdb_results.png"><img src="https://static.igem.org/mediawiki/2010/6/63/Ccdb_results.png" class="shadow" style="float:center;width:850px;margin:10px"></a></html></p><br />
<p>&nbsp;</p><br />
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<br />
<p>'''Vectors Used:'''</p><html><a href="https://static.igem.org/mediawiki/2010/f/f4/Vectors_ccdB.png"><img src="https://static.igem.org/mediawiki/2010/f/f4/Vectors_ccdB.png" class="shadow" style="float:left;width:500px;margin:10px"></a></html><br />
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!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/CompositeTeam:Nevada/Composite2010-10-27T19:22:10Z<p>Ebersaba: </p>
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<p>&nbsp;</p><br />
== Composite Parts ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*RD29A Promoter + Strong Plant Kozak (RBS) + RFP [[Team:Nevada/registry submissions]]<br />
*35S Promoter + (Strong Plant RBS + GFP) From K414001 [[Team:Nevada/registry submissions]]<br />
----<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">rd29A – RFP</span>: This composite is designed so that transformed plants will yield red fluorescent protein under cold, drought, or high soil salinity conditions. We were successful in developing cold-responsive red fluorescent NT cells in time for the jamboree. Please check out the <html><a href="https://2010.igem.org/Team:Nevada/Results"><span style="color:#1569C7;font-weight:bold;">Transformation Results</span></a></html>.</p><br />
For more information on rd29A, click <html><a href="https://2010.igem.org/Team:Nevada/RD29A"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>.<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">35S – GFP</span>: This composite is designed so that transformed plants will have constitutive expression of green fluorescent protein. Having this constant expression provides the basis to measure any other inducible fluorescent proteins’ fluorescence relative to an internal control. </p><br />
For more information on 35S, click <html><a href="https://2010.igem.org/Team:Nevada/35S"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>.<br />
<p>&nbsp;</p><br />
<p><span style="text-decoration:underline;font-weight:bold;">DREB1C - EYFP</span>: Although not developed in time for this year's iGEM competition. We had planned on developing a composite part that would respond specifically to cold response. This would provide a specific signal and not be as receptive to various stresses like RD29A. Under cold stress, this composite would express enhanced yellow fluorescent protein.</p> <br />
For more information on DREB1C, click <html><a href="https://2010.igem.org/Team:Nevada/DREB1C"><span style="color:#1569C7;font-weight:bold;">here</span></a></html>. <br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/plasmid_w_terminator_sequencesTeam:Nevada/plasmid w terminator sequences2010-10-27T19:21:53Z<p>Ebersaba: </p>
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<p>&nbsp;</p><br />
== Plasmid with Terminator Sequence ==<br />
<br />
<p>'''piGEM10 Plasmid Construction'''<br />
<br>Plant transformation vector piGEM10 was derived from the vector pBIB HYG (Becker (1990) Nuc Acid Res 18: 203). To construct this plasmid, a synthetic gene containing the Arabidopsis stress induced RD29 promoter, a red fluorescent protein (RFP) containing a plant optimized ribosome binding site, and the nopoline synthase (NOS) 3’ plant specific transcriptional terminator. The NOS terminator contains a plant specific sequences that signals transcriptional termination and the addition of a polyadenyled 3’ tail to the mature mRNA. HinD3, EcoR1 and Xba1 restriction sites were added respectively to the 5’ end of the RD29 promoter. Spe1 and Pst1 sites were inserted between the stop codon of the RFP gene and the NOS terminator. Additionally, an Mfe1 site was added to the 3’ end of the NOS 3’ terminator. This synthetic gene was subcloned into a plasmid designated as piGEM Nevada.</p><br />
<br><br />
<p>The synthetic gene construct from piGEM Nevada was then subcloned as a HinD3 Mfe1 fragment into the HinD3 EcoR1 site of pBIB HYG. Because Mfe1 and EcoR1 restriction sites generate compatible sticky ends that when joined cannot be re-cleaved by either enzyme, by subcloning the synthetic gene as a HinD3 Mfe1 fragment instead of a HinD3 EcoR1 fragment we were able to destroy the EcoR1 site located at the 3’ end of the NOS 3’ terminator. The resulting plasmid now contains Biobrick compatible cut sites flanking the 5’ end of the promoter and 3’ end of the reporter gene. This configuration will allow for the exchange of the RD29 promoter / RFP fusion with any other promoter / gene fusion construct. We are in the process of removing an unwanted Pst1 site located in the plasmid backbone. However, the vector is still useable now by cloning promoter / gene fusions as EcoR1 Spe1 fragments into the EcoR1 Spe1 sites of piGEM10.</p><br />
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<html><a href="https://static.igem.org/mediawiki/2010/0/05/PiGEM10.gif"><img src="https://static.igem.org/mediawiki/2010/0/05/PiGEM10.gif" class="shadow" style="float:left;width:900px;margin:10px"></a></html> <br />
<br><br />
<br><p>pBIB HYG was chosen as the basis for our vector construction based on the following criteria. First, unlike many plant transformation vectors, pBIB HYG appears to be freely available in the public domain. Patent searches failed to turn up any past or pending patents that may restrict the use of the plasmid. Second, pBIB HYG is derived from the well characterized pBIN19 plant transformation vector series which has been used in the transformation of a wide variety of plant species. Third, pBIB HYG required the fewest modifications to become compatible with current Biobrick standards.<br />
<br><br />
<br>'''piGEM10 Features'''<br />
<br>The piGEM10 backbone, which is derived from pBIN19, possesses a RK2 broad host plasmid origin of replication that allows for its use in both E. coli and Agrobacterium tumefaciens. The RK2 origin of replication allows the investigator to manipulate the plasmid in E. coli and then transform the finished plasmid to the appropriate Agrobacterium strain for transformation into plants. One unfortunate consequence of plasmids with RK2 origins is that they are low copy number plasmids and therefore one must use larger cultures when isolating these plasmids. piGEM10 also possesses a bacterial expressed neomycin phosphotransferase 2 (NPT2) gene that confers kanamycin resistance in bacteria carrying this plasmid. 50 μg/mL of kanamycin should be used to select for bacterial transformants containing this plasmid. The plasmid also contains the T-DNA Left (LB) and Right Border (RB) sequences which are required for the integration of recombinant DNA fragments into the genome of target plant transformation host. Between the LB and RB are two plant gene expression cassettes. The first expression cassette consist of the nopoline synthase promoter, the hygromycin B phosphotransferase (HPT) gene and a plant polyadenylation signal sequences. The nopoline synthase promoter allows for high-level and constitutive transcription HPT in plant cells. When selecting for plants transformed with this vector, 50 μg/mL hygromycin should be added to the tissue culture medium. The second gene cassette consists of the plant stress inducible RD29 promoter; a plant optimized red fluorescent protein gene and nopoline synthase NOS terminator.</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/long_term_goalsTeam:Nevada/long term goals2010-10-27T19:21:19Z<p>Ebersaba: </p>
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== Long Term Goals ==<br />
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<p><br />
*To promote the use of plant systems as biosensors.<br />
*To promote public knowledge concerning genetic engineering and synthetic biology.<br />
*To develop a library of plant-specific stress-inducible promoters for future iGEM competitions.<br />
*To address logistical hurdles in utilizing various reporter genes in plant biosensor applications.<br />
*Investigate potential plant systems for utilization in remote-sensing applications.</p><br />
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'''Demonstrating that plant systems can be used as biosensors is well and good, but several issues need to be addressed before implementation of plant biosensors can occur:'''<br />
<br><br />
<br><br />
1. An energy/cost efficient infrastructure for the detection of reporter genes in crop plants must be developed. One of the main problems in using fluorescent proteins as reporters for plant biosensors arises due to autofluorescent properties present in compounds throughout plants cells. Chlorophyll fluorescence has already been used in previous studies to measure temperature stress in plants, so necessary filtering mechanisms must be implemented to discern differences between reporter fluorescence and the natural autofluorescent properties in plants (Chaerle et al.). Therefore, it may be prudent to look into reporter genes other than those that code for fluorescent proteins for implementation in plant biosensors.<br />
<br><br />
<br><br />
2. If a fluorescent or bioluminescent reporter system was to be implemented in crop plants, how exactly will stress-dependent reporter activity be detected? Satellite monitoring is a possibility, but this could potentially only be performed during the night to avoid detection complications due to sunlight (Chaerle et al).<br />
<br><br />
<br><br />
3. Should crop plants be engineered with built-in biosensing capabilities, or should plants other than crop plants be grown in parallel to act as biosensors? It would probably make the most sense to grow the biosensor capable crop species of interest in parallel with the crop to control for any disparities in life cycle length. <br />
<br><br />
<br><br />
4. Once a plant biosensor system has been proven to be useful the system must be deemed as safe by regulatory bodies such as the USDA and FDA.<br />
<br><br />
<br><br />
'''References'''<br />
<br><br />
'''Chaerle et al.''' Monitoring and screening plant populations with combined thermal and chlorophyll fluorescence imaging. Journal of Exp Botany., 58: 773-784, 2010.<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/plants_as_remote_sensorsTeam:Nevada/plants as remote sensors2010-10-27T19:21:03Z<p>Ebersaba: </p>
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==Plants as Remote Sensors==<br />
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<p>The passage of “The Stockholm Convention on Persistant Organic Pollutants” in 2008 clearly demonstrates that the world has taken a much more active approach in addressing concerns about the environmental and ecological impact of pollutants (Rodriguez-Mozaz et al, 2006). By demonstrating the practicality of plants as model biosensors for remote sensing, the Nevada team has addresses several issues that have plagued the field of biosensors since their original conception. First by utilizing a natural receptor that can be localized to a specific cell type in plants, Nevada has shown the usefulness of transgenic plant biosensors that will respond locally to toxic compounds. In addition, natural biosensors that do not inhibit basic metabolic functions and only fluoresce allow for a simpler model system that is easily adapted for future uses. Secondly, model plant organisms are much larger reporters of environmental stresses and can be easily clustered together to form ‘reporter clusters’ that are easily viewed at the source of the contamination. Finally, the natural life cycle of plant organisms (when compared to traditional microorganism biosensors) is much longer and allows for the continued monitoring of a contaminated area over a much longer time while still providing a feasible option to control the plant’s reproductive cycle. As discussed in “Perspective on Optical Biosensors and Integrated Sensor Systems” (Ligler, 2009), the use of on-site optical biosensors to tackle complex and difficult issues like environmental monitoring, food testing, and even counter- terrorism are driving issues in the proliferation of remote sensing technology.</p><br />
<br><br />
<p>Despite these potential successes, challenges still remain when utilizing and applying biosensors to environmental concerns. Societal concerns about contamination with wild type plants are an ongoing and legitimate concern. It may be possible to eliminate such concerns by engineering in a sterility defect that is coupled with a fluorescent reporter, it may be possible to prevent transgenic contamination. For example, utilizing satellites to image biosensors from space would require a different, possibly IR receptor that can be viewed at great distances. Despite concerns for practicality and issues of feasibility, by simply demonstrating that biosensors in plants is possible, further research is required to determine how advantageous plant biosensors are when weighing the concerns for successfully remediating the environment.<br />
<br><br />
<br>'''References'''<br />
<br>'''Rodriguez-Mozaz et al.''', 2006, Analytical. Bioanal Chem. 386: 1025-1041<br />
<br>'''Ligler, F.S.''', 2009, Analytical. Chem. 81: 519-526</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/plant_compatible_reportersTeam:Nevada/plant compatible reporters2010-10-27T19:20:33Z<p>Ebersaba: </p>
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== Reporters ==<br />
https://static.igem.org/mediawiki/2010/9/98/Finished_final.png<br />
*Strong Plant RBS (Kozak sequence) + GFP from E0040 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + EYFP from E0030 [[Team:Nevada/registry submissions]]<br />
*Strong Plant RBS (Kozak sequence) + mCherry from J06504 [[Team:Nevada/registry submissions]]<br />
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<html><br />
<a href="https://static.igem.org/mediawiki/2010/7/7e/Reporters.png"><img src="https://static.igem.org/mediawiki/2010/7/7e/Reporters.png" class="shadow" style="float:left;width:450px;margin:10px"></a><br />
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<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Building Consensus</span></p><br />
<br />
While all the aforementioned issues are important, the aspect of plant engineering that we believe is fundamental for future iGEM teams to consider is the ribosome binding site (RBS). RBS can differ between species, but it varies widely between eukaryotes, such as yeast, animals, and plants. The term RBS can be misleading because ribosomes can weakly associate with RNA as it “scans” along the sequence. Why is the ribosome “scanning?” Ribosomes initiate translation, and the “start” site that we have all been taught for eukaryotes is the methionine sequence AUG (or ATG if you are biased towards DNA). However, almost thirty years ago, a researcher named Kozak discovered that it is not simply AUG which initiates translation but the context of that AUG, the surrounding sequence, influenced whether translation actually began with one AUG sequence versus another. These context sequences, as they have been discovered in different organisms, eventually have been named Kozak sequences. <br />
<br />
Even among plants, there can be different Kozak sequences. Where we decided to contribute to iGEM was to supply the registry with the first fluorescent proteins with plant compatible RBS or Kozak sequences. We have chosen a generically ‘strong’ Kozak sequence that should provide the maximum translational efficiency for dicots, but it should also work generally well enough in most if not all plants. Our sequence is AAA AAA AAA ACA upstream of the AUG. An important aspect of Kozak sequences one should consider is there are both an upstream component and a downstream component. The string of purines upstream is associated with many plant Kozak sequences, but almost equally important is to have a G at the +4 position, or immediately following the AUG. Therefore, AAAAAAAAACA'''AUG'''G is likely to have the highest translational efficiency. Fortunately, two of the florescent proteins, EYFP and mCherry have this context. GFP, however does not. It is missing the G at +4 which will hurt its translational efficiency. Instead, a C occupies that position which codes for arginine, R. There is no codon for arginine that starts with G. Unless a known mutation can be made, we may stuck with that hindrance. However, we have attempted to compensate in one of our composite parts, 35S GFP. 35S is a constitutive plant promoter. Ideally, the high transcriptional activity can compensate for the weakened translational efficiency. <br />
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<br />
----<br />
<p style="text-align:center;"><span style="text-decoration:underline;font-weight:bold">Engineering Possibilities (Fine tuning your translation)</span></p><br />
As we see it, there are three ways future iGEM teams could engage the plant Kozak sequence to modify gene expression in plants: identity, distance, or deking.<br />
<br />
'''1) Identity''': The most obvious way of affecting translational efficiency would be to alter the Kozak sequences. Having genes each prefaced with the same promoter but with different Kozak contextual sequences would tier the levels of expression. One could have an optimum Kozak like the one we have submitted and also engineer a weaker Kozak sequence for another gene which has relatively 50% expression compared to the optimum gene expressed. Consulting literature or experimenting in less-frequently researched plants will allow for greater variability in controlling expression. <br />
<br />
'''2) Distance''': Another way to affect your protein expression would be how far the ‘true’ Kozak sequence is relative to the 5’ cap. A strong Kozak sequence means nothing if it is several hundred base pairs away from the end of the promoter. Because our team wanted to supply genes with maximal expression, our parts are intended to be placed immediately behind the promoter. Yet, engineering plasmids that put gaps between the promoter and actual Kozak, or primers designed to put more space in between the promoter and start site, could also be one way of dialing the levels of expression. <br />
<br />
'''3) Deking''':(Realizing a team from a desert is using a hockey term): The “fake out.” A third alternative that combines the principles of identity and distance is to create one, two, or a few pseudo-start sites. Psuedo-start sites means one would engineer AUG sequences upstream of the actual, desired one. These sequences would be in a poorer context and/or would translate into little nonsense peptides that theoretically have no function. Think of them as siAUG (short interfering AUG sites). These fake sites would knockdown expression. <br />
<br />
In Summary, Kozak sequences have plenty of promise in the engineering side of iGEM, but Kozak sequences are also a necessity that all iGEM teams must consider if they are to express proteins in plants.<br />
<br />
<span style="text-decoration:underline;font-weight:bold">References</span><br />
<br />
'''Agarwal, S., Jha, S., Sanyal, I., Amla, D.V.''' (2009) Effect of point mutations in translation initiation context on the expression of recombinant human alpha1-proteinase inhibitor in transgenic tomato plants. ''Plant Cell Reports''. 28: 1791-1798.<br />
<br><br />
'''Joshi, C.P., Zhou, H., Huang, X., Chiang, V.L.''' (1997) Context sequences of translation initiation codon in plants. ''Plant Molecular Biology''. 35: 993-1001.<br />
<br><br />
'''Kozak, M.''' (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribososomes. ''Cell''. 44: 283-92.<br />
<br><br />
'''Matsuda, D., Dreher, T.W.''' (2006) Close spacing of AUG initiation codons confers dicistronic character on a eukaryotic mRNA. ''RNA''. 12: 1138-1349.<br />
<br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/promotersTeam:Nevada/promoters2010-10-27T19:20:05Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Promoters UNR Final.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
== Promoters ==<br />
<br />
<br />
<br />
<br />
<html><br />
<div id="vertmenu"> <br />
<h1>Subpages</h1><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Nevada/DREB1C" tabindex="1">DREB1C</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/RD29A" tabindex="2">rd29A</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/35S" tabindex="3">35S</a></li><br />
<li><a href="https://2010.igem.org/Team:Nevada/CD2Inducible" tabindex="4">CD2+ Inducible</a></li><br />
</ul><br />
</div><br />
</html><br />
<br />
<html><br />
<body link=#00ffff><br />
<a href="https://static.igem.org/mediawiki/2010/7/79/GFP_tobacco_plant.jpg"><img src="https://static.igem.org/mediawiki/2010/7/79/GFP_tobacco_plant.jpg" class="shadow" style="float:left;width:170px;margin:10px"></a><br />
</html><p>The Nevada team has created environmental stress sensors by using <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter"><span style="color:#1569C7;font-weight:bold;">rd29A</span></a></html> and <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> promoters to express red fluorescent protein and other bio-fluorescent markers. When induced by environmental stress, plants carrying these genes can easily be detected by the farmer walking through his field or by a plane flying over acres of farmland. Promoter elements are short and easy to work with. They also allow for modification and specialization. <html><a href="https://2010.igem.org/Team:Nevada/DREB1CPromoter"><span style="color:#1569C7;font-weight:bold;">DREB1C</span></a></html> and <html><a href="https://2010.igem.org/Team:Nevada/RD29APromoter"><span style="color:#1569C7;font-weight:bold;">rd29A</span></a></html> both have multiple binding motifs in their promoter regions allowing for variation in expression levels and the particular stresses that induce them. 35S is a constitutive promoter that can be valuable for control groups in stress and other plant response research.</p><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<p>Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</p><br />
<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/TeamTeam:Nevada/Team2010-10-27T19:19:17Z<p>Ebersaba: </p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:Picture Team.png|border|left|950px]]<br />
<br />
{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span>&nbsp;<span style="font-family:Arial;font-size:16pt;text-decoration:bold;color:#15317E;background-color:ffffff;">Meet the Nevada Team</span>&nbsp;<span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&diams;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&clubs;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#E41B17;">&hearts;</span><span style="font-family:Arial;font-size:18pt;text-decoration:bold;color:#000000;">&spades;</span>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<br />
----<br />
<div style="padding: 10px 0 10px 10px;"><br />
<br />
<html><br />
<a href="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b9/IMG_5969.JPG/600px-IMG_5969.JPG" class="shadow" style="float:left;margin:0px;width:600px"></a><br />
</html><br />
<br />
<p style="text-align:center;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Advisors</span><br />
</p><br />
----<br />
<br />
[[Image:Christie.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. Christie Howard</span></p>]]<br />
<br />
[[Image:Shintani.jpg|center|thumb|alt=alt text|<p style="text-align:center;"><span style="font-family:Times;font-size:12pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Dr. David Shintani</span></p>]]<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:none;text-decoration:bold;color:#15317E;background-color:ffffff;">Undergraduates</span><br />
</p><br />
----<br />
<gallery widths=170px heights=170px perrow=4><br />
Image:Hilary UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span></p><br />
Image:mpolasko2.JPG|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span></p><br />
Image:Randy.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span></p><br />
Image:Copley.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span></p><br />
Image:Bryson UNR 2.png| <p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span></p><br />
Image:Gladwill.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span></p><br />
Image:Image-Elaine.jpeg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span></p><br />
Image:Sam final UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span></p><br />
&nbsp;<br />
Image:Nick UNR.png|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span></p><br />
Image:Hileary.jpg|<p style="text-align:center;"><span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span></p><br />
</gallery><br />
<br />
<br />
<p style="text-align:Left;"><span style="font-family:Arial;font-size:16pt;font-style:normal;font-weight:normal;text-decoration:bold;color:#15317E;background-color:ffffff;">What We Did</span><br />
</p><br />
----<br />
<br />
<div style="padding:10px;clear:both"><br />
{| border="0" cellpadding="0" cellspacing="20"<br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Hilary Allen</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Elaine Bersaba</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Matthew Polasko</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Christian Copley</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Team President</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">NT Cell Transformations</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter + Plant Kozak + RFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + EYFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Plant Kozak + mCherry</span><br />
*<span style="font-size:12pt;font-weight:bold;">Composite - 35s Promoter + Plant Kozak + GFP</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;"> DREB1C Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Website Design</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Bryson Wheeler</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Richard Hilleary</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Nick Noel</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Samantha Lee</span><br />
|-<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">35S Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">Modeling</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">Fundraising</span><br />
*<span style="font-size:12pt;font-weight:bold;">Cadmium Inducible Promoter</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">RD29A Promoter</span><br />
*<span style="font-size:12pt;font-weight:bold;">RD29A transformation construct</span><br />
|-<br />
|&nbsp;<br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Randy Pares</span><br />
|<span style="font-family:Times;font-size:14pt;font-style:normal;font-weight:bold;text-decoration:none;color:#15317E;background-color:ffffff;">Vadim Gladwill</span><br />
|-<br />
|&nbsp;<br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|<br />
*<span style="font-size:12pt;font-weight:bold;">ccdB</span><br />
|}<br />
----<br />
<br />
</div><br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/File:Sda_logo_small.jpgFile:Sda logo small.jpg2010-10-27T19:18:09Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T19:18:01Z<p>Ebersaba: </p>
<hr />
<div>{{nevadamain}}<br />
== Abstract ==<br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
</html><br />
<br />
<br><br />
<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, the''' 2010 Nevada iGEM Team''' has three goals for this year’s competition. First, we are going to test the validity of utilizing Nicotiana tabacum (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. These cells can therefore be utilized as a quick proof-of-concept test model before moving synthetic constructs into plants of interest. We also aim to produce an iGEM-compatible plant-specific plasmid, several stress-inducible plant promoters, reporter genes containing Kozak sequences (ribosome binding sites) and terminators that conform to BioBrick standards. Lastly, we hope to measure the induction of these stress promoters in real-time by performing a fluorometry assay in which stress will be applied to NT cells and fluorescent output by a reporter (GFP) will be measured to detail induction in real time. This method has a distinct advantage over microarrays since microarrays are only one ‘snapshot’ in time.</p><br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo small.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/File:Sda_logo.jpgFile:Sda logo.jpg2010-10-27T19:16:04Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T19:15:52Z<p>Ebersaba: </p>
<hr />
<div>{{nevadamain}}<br />
== Abstract ==<br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
</html><br />
<br />
<br><br />
<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, the''' 2010 Nevada iGEM Team''' has three goals for this year’s competition. First, we are going to test the validity of utilizing Nicotiana tabacum (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. These cells can therefore be utilized as a quick proof-of-concept test model before moving synthetic constructs into plants of interest. We also aim to produce an iGEM-compatible plant-specific plasmid, several stress-inducible plant promoters, reporter genes containing Kozak sequences (ribosome binding sites) and terminators that conform to BioBrick standards. Lastly, we hope to measure the induction of these stress promoters in real-time by performing a fluorometry assay in which stress will be applied to NT cells and fluorescent output by a reporter (GFP) will be measured to detail induction in real time. This method has a distinct advantage over microarrays since microarrays are only one ‘snapshot’ in time.</p><br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
</html><br />
<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Sda logo.jpg]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:NevadaTeam:Nevada2010-10-27T19:13:16Z<p>Ebersaba: </p>
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<div>{{nevadamain}}<br />
== Abstract ==<br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/thumb/8/8b/IGEM_Team_2010.jpg/400px-IGEM_Team_2010.jpg" class="shadow" style="float:right"><br />
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<br />
<br><br />
<p>Our long term goals are to develop plant biosensors that reliably measure changes in the local environment. In the process of developing biosensors, the''' 2010 Nevada iGEM Team''' need to develop fundamental promoters, reporters, and plasmids for plants. Therefore, the''' 2010 Nevada iGEM Team''' has three goals for this year’s competition. First, we are going to test the validity of utilizing Nicotiana tabacum (NT cells) as a model for the expression of higher plant genes for future iGEM competitions. NT cells are a faster, cheaper, and safer model than traditional plant transformation. These cells can therefore be utilized as a quick proof-of-concept test model before moving synthetic constructs into plants of interest. We also aim to produce an iGEM-compatible plant-specific plasmid, several stress-inducible plant promoters, reporter genes containing Kozak sequences (ribosome binding sites) and terminators that conform to BioBrick standards. Lastly, we hope to measure the induction of these stress promoters in real-time by performing a fluorometry assay in which stress will be applied to NT cells and fluorescent output by a reporter (GFP) will be measured to detail induction in real time. This method has a distinct advantage over microarrays since microarrays are only one ‘snapshot’ in time.</p><br />
<br><br />
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png"><img src="https://static.igem.org/mediawiki/2010/9/9c/Theory_Home-foot_UNR.png" class="shadow" style="float:left;width:900px;margin:10px"></a><br />
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<p style="text-align:right;"><span style="font-size:10px;">Fluorescent Plant Image Taken From: http://www.edinformatics.com/inventions_inventors/genetic_engineering.htm</span></p><br />
<br />
----<br />
<br />
'''We would like to thank the following sponsors for their support in helping us make this project possible.'''<br />
Much thanks to the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Majors.aspx Departments of Biochemistry and Biotechnology]</span> and the <span style="font-weight:bold;">[http://www.cabnr.unr.edu/Students/Glance.aspx College of Agriculture, Biotechnology and Natural Resources]</span> for their encouragement and support. Thank you <span style="font-weight:bold;">[http://www.unr.edu/inbre/ Nevada INBRE]</span> for over $16,000 in support for supplies and registration costs.<br />
Thank you to <span style="font-weight:bold;">[https://asun.unr.edu/Default.aspx Associated Students of the Univeristy of Nevada]</span> for supporting our fund raising efforts.<br />
Thank you to <span style="font-weight:bold;">[http://www.promega.com/Catalog/CountrySelect.aspx?returnurl=/Default.asp Promega Co.]</span> for free enzyme donations.<br />
Thank you to <span style="font-weight:bold;">[http://www.invitrogen.com/site/us/en/home.html?cid=covinvggl89100000002336s& Invitrogen Co.]</span> for a discount on our Vector NTI program.<br />
{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
!align="center"|[[Image:Logo.gif]]<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T19:01:58Z<p>Ebersaba: /* AUGUST */</p>
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<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
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<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR 1 & Pst 1 Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
<p>[[Image:GFP from iGEM (E0040) paint.jpg|500px]]</p><br />
* The "GFP E0040" gel analysis was successful for the expected bands of GFP showed at ~720 bp when digested with EcoR 1 & Pst 1 (labeled Cut #1, Cut #2, Cut#3, Cut#4).<br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
<p>[[Image:GFP PCR product.jpg|500px]]</p><br />
* The "GFP PCR" gel analysis was successful for the expected bands of GFP + Primers showed at ~ 804bp for all PCR products (labeled 1 ng, 5ng, 10ng, & ~826ng)<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p><br />
* The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp. <br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony #17 (Digested/Cut sample labeled C17) the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony #17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T18:59:40Z<p>Ebersaba: /* JULY */</p>
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
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==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR 1 & Pst 1 Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
<p>[[Image:GFP from iGEM (E0040) paint.jpg|500px]]</p><br />
* The "GFP E0040" gel analysis was successful for the expected bands of GFP showed at ~720 bp when digested with EcoR 1 & Pst 1 (labeled Cut #1, Cut #2, Cut#3, Cut#4).<br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
<p>[[Image:GFP PCR product.jpg|500px]]</p><br />
* The "GFP PCR" gel analysis was successful for the expected bands of GFP + Primers showed at ~ 804bp for all PCR products (labeled 1 ng, 5ng, 10ng, & ~826ng)<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p><br />
* The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp. <br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony # 17 (Digested/Cut sample salbeled C17) tha the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/File:GFP_from_iGEM_(E0040)_paint.jpgFile:GFP from iGEM (E0040) paint.jpg2010-10-27T05:22:06Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T05:21:48Z<p>Ebersaba: /* JULY */</p>
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<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
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<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
<p>[[Image:GFP from iGEM (E0040) paint.jpg|500px]]</p><br />
* The "GFP E0040" gel analysis was successful for the expected bands of GFP showed at ~720 bp when digested with EcoR 1 & Pst 1 (labeled Cut #1, Cut #2, Cut#3, Cut#4).<br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
<p>[[Image:GFP PCR product.jpg|500px]]</p><br />
* The "GFP PCR" gel analysis was successful for the expected bands of GFP + Primers showed at ~ 804bp for all PCR products (labeled 1 ng, 5ng, 10ng, & ~826ng)<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p><br />
* The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp. <br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony # 17 (Digested/Cut sample salbeled C17) tha the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
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!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
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!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/File:GFP_PCR_product.jpgFile:GFP PCR product.jpg2010-10-27T05:12:11Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T05:11:48Z<p>Ebersaba: /* AUGUST */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
<p>[[Image:GFP PCR product.jpg|500px]]</p><br />
* The "GFP PCR" gel analysis was successful for the expected bands of GFP + Primers showed at ~ 804bp for all PCR products (labeled 1 ng, 5ng, 10ng, & ~826ng)<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p><br />
* The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp. <br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony # 17 (Digested/Cut sample salbeled C17) tha the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/File:GFP_in_Topo_Vector_001.jpgFile:GFP in Topo Vector 001.jpg2010-10-27T05:06:26Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T05:06:11Z<p>Ebersaba: /* AUGUST */</p>
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<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
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<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p><br />
* The "GFP in Topo Vector" gel analysis was successful for the EcoR1 digested (cut samples labeled C1, C2, C3, C4, C5, & C6) samples shows bands at ~804bp (GFP + primers) and the Topo bands at ~ 3,519bp. <br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony # 17 (Digested/Cut sample salbeled C17) tha the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
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!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
<br />
|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T05:02:21Z<p>Ebersaba: /* AUGUST */</p>
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[[Image:UNR Notebook.png|border|left|950px]]<br />
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<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony # 17 (Digested/Cut sample salbeled C17) tha the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
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!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T05:01:47Z<p>Ebersaba: /* SEPTEMBER */</p>
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<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
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==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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{| style="color:#FFFFFF;background-color:#FFFFFF;" cellpadding="3" cellspacing="1" border="1" bordercolor="#008000" width="100%" align="center"<br />
!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
!align="center"|[[Image:NV_INBRE_Logo.jpg|200px]]<br />
!align="center"|[[Image:UNR_ASUN_logo.jpg]]<br />
!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/File:GFP_in_pSB1C3_iGEM_Vector.jpgFile:GFP in pSB1C3 iGEM Vector.jpg2010-10-27T04:56:37Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T04:55:24Z<p>Ebersaba: /* SEPTEMBER */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony # 17 (Digested/Cut sample salbeled C17) tha the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
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!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T04:53:09Z<p>Ebersaba: /* SEPTEMBER */</p>
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<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "GFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed at colony # 17 (Digested/Cut sample salbeled C17) tha the GFP (720) + primers [which includes the KpnI (GGT ACC = 6bp), Kozak sequence (AAA AAA AAA ACA = 12bp), & Translationsal Stop codon (TAATAA = 6bp)] band is at ~804bp and the pSB1C3 bands at ~2,072bp. <br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T04:20:35Z<p>Ebersaba: /* OCTOBER */</p>
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
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==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique and made sure I streaked the 45 single colonies on LB-Chloramphenicol plates. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate. <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
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!align="center"|[[Image:Promega_logo.jpg]]<br />
!align="center"|[[Image:Invitrogen_logo.jpeg]]<br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T04:17:32Z<p>Ebersaba: /* SEPTEMBER */</p>
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<div>{{Nevada_css}}<br />
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<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4). <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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|}</div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T04:16:47Z<p>Ebersaba: /* SEPTEMBER */</p>
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
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==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p><br />
* The "rd29A promoter + RFP in pSB1C3 iGEM Vector" gel analysis was successful for it showed the rd29A + RFP bands at ~1,529 bp and the pSB1C3 bands at ~2,072bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C1-2, C1-7, C1-9).<br />
<br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4). <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
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!align="center"|[[Image:Nevada_CABNR.jpg|200px]]<br />
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|}</div>Ebersabahttp://2010.igem.org/File:Rd29A_promoter_%2B_RFP_in_pSB1C3_iGEM_Vector.jpgFile:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg2010-10-27T04:12:57Z<p>Ebersaba: </p>
<hr />
<div></div>Ebersabahttp://2010.igem.org/Team:Nevada/NotebookTeam:Nevada/Notebook2010-10-27T04:07:19Z<p>Ebersaba: /* OCTOBER */</p>
<hr />
<div>{{Nevada_css}}<br />
[[Image:UNR Notebook.png|border|left|950px]]<br />
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{{Nevada_topbar}}<br />
<div style="padding: 10px 10px 30px 10px;"><br />
<br />
<p>&nbsp;</p><br />
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<p style="text-align:left;"><span style="font-family:Arial;font-size:16pt;font-style:italic;font-weight:none;text-decoration:italic;color:#15317E;background-color:ffffff;">Team Nevada Notebook</span><br />
</p><br />
----<br />
==APRIL==<br />
'''Week of April 11-17:'''<br />
<br />
* ''Christian''<br />
**Transformed pBIB into Top 10 Cells<br />
<br />
* ''Bryson, Michael, Senny, Tyler''<br />
** Made tobacco cell (NT-1) media in Dr. Shintani's lab<br />
<br />
'''Week of April 18-24:'''<br />
<br />
* ''Bryson, Christian'' <br />
** EcoRI digest of pBIB<br />
** Made Na acetate buffer<br />
* ''Christian''<br />
** Ran gel of EcoR1 Digested pBIB<br />
==MAY==<br />
'''Week of April 25-May 1:'''<br />
<br />
* ''Bryson''<br />
** Ran agarose gel of EcoRI digest<br />
<br />
* ''Matthew''<br />
** Team collaborated on pBIB assignment. Too many chefs in the kitchen.<br />
** We decide to each tackle the pBIB problem in parallel<br />
<br />
'''Week of May 2-8:'''<br />
<br />
* ''Elaine''<br />
** Ran 0.8% agarose gel of EcoRI digest<br />
** Made LB/KAN plates<br />
** Spread for colonies<br />
** Did miniprep for pBIB liquid cultures<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digest and Klenow<br />
** Ethanol precipitated and ligated<br />
<br />
'''Week of May 9-15:'''<br />
<br />
* ''Christian''<br />
** EcoR1/Xba1 Digest of pBIB<br />
** Did Mini prep on 4 cultures<br />
<br />
* ''Bryson'' <br />
** Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.<br />
** Did minipreps on additional pBIB liquid cultures.<br />
<br />
* ''Elaine''<br />
** Did XbaI and EcoRI digest of pBIB<br />
** Ran 2 0.8% gels of each digest<br />
** Did a miniprep and a Phenol:chloroform clean-up<br />
** Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB<br />
<br />
* ''Matthew''<br />
** Ligated and transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 16-22:'''<br />
<br />
* ''Christian''<br />
** Generated glycerol stock of pBIB transformed Top 10 cells<br />
** Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates<br />
** Ran samples on gel after digest with EcoR1<br />
<br />
* ''Bryson''<br />
** Klenow reactions of EcoRI digests<br />
** Phenol:chloroform cleanup of pBIB prior to ligation<br />
** Blunt-end ligation of klenowed pBIB<br />
<br />
* ''Randy Pares and Vidim Gladwell''<br />
** Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.<br />
<br />
* ''Elaine''<br />
** Made LB/KAN plates<br />
** Made 50mg/ml stock of KAN<br />
** Made 1X TAE buffer<br />
<br />
* ''Matthew''<br />
** Digested and tested more colonies, none worked<br />
** Started over with pBIB and Klenow<br />
** Ethanol precipitated and ligated<br />
** Transformed<br />
** Screened colonies, miniprepped, and digested, ran on gel<br />
** No candidates had EcoRI eliminated<br />
<br />
'''Week of May 23-29:'''<br />
<br />
* ''Christian''<br />
** Miniprep on pBIB transformed Top 10 cells using "low copy #" modification<br />
** Digested with EcoR1 and HindIII<br />
<br />
* ''Bryson''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (samples 3).<br />
** Miniprepped sample 3, using modified protocol for large/low-copy plasmids.<br />
** EcoRI digest of uncut sample 3<br />
** Prepared 5 mM dNTP stock<br />
<br />
* ''Elaine''<br />
** Incubated 50 mL liquid culture of ''E. coli'' with pBIB (sample 4 & sample 5)<br />
** Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids<br />
** Nanodrop of DNA recovery of miniprepped sample 4 & sample 5<br />
<br />
* ''Randy and Vadim''<br />
** Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22<br />
** Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Investigated alternative ways of eliminating EcoRI site. <br />
** Want to try hexameric linkers<br />
<br />
==JUNE==<br />
'''Week of May 30-June 5:'''<br />
<br />
* ''Christian''<br />
** Miniprep Top 10 Cells transformed with pBIB<br />
<br />
* ''Bryson''<br />
** HinD3 digest of uncut sample 3<br />
** Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI<br />
** Pooled uncut samples 3, 4 and 5 (pBIB-pool)<br />
** Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)<br />
** EcoRI digest of pBIB-pool and pBIB-maxi<br />
** Ran 0.8% agarose gel of EcoRI digests<br />
** Klenow reactions of pBIB-pool and pBIB-maxi<br />
** Made glycerol stocks of pBIB samples 1-5<br />
<br />
* ''Elaine''<br />
** EcoRI digest of uncut pBIB sample 4 and 5<br />
** HinD3 digest of uncut sample 4 & sample 5 as a positive control<br />
** Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful<br />
** Ethanol precipitation of the EcoRI digests of sample 4 & sample 5<br />
** Nanodrop resulted to a low DNA content<br />
** Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep<br />
** Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest <br />
** Modified all protocols of the Binary vector <br />
<br />
* ''Randy and Vadim''<br />
** Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)<br />
** Programmed thermal cycler for PCR of ccdB gene<br />
** Ran PCR for ccdB<br />
** Prepared LB/KAN Broth<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel<br />
*** Reaction was unsuccessful<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran EcoRI digests<br />
** Ran ethanol precipitation<br />
** Ligated with several concentrations of hexameric linkers designed to destroy site<br />
** Ran transformation, no colonies<br />
*** Believed procedures/conditions ran on ligation and transformation not ideal<br />
<br />
'''Week of June 6-12:'''<br />
<br />
* ''Christian''<br />
** Klenow reaction on EcoR1 digested pBIB<br />
** T4 ligation on Klenow products<br />
<br />
* ''Bryson''<br />
** Ligation reactions for pBIB-pool and pBIB-maxi<br />
** Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB<br />
** Transformed Top-10 Cells with modified pBIB (designated pBIB#)<br />
*** Obtained two colonies after overnight incubation<br />
** Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate <br />
<br />
* ''Randy and Vadim''<br />
** Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI<br />
** Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB<br />
** Modified thermal cycler conditions for PCR of ccdB gene<br />
** Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)<br />
** Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene<br />
<br />
https://static.igem.org/mediawiki/2010/7/76/PCR_ccdB_confirmation.png<br />
<br />
** Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit<br />
<br />
* ''Matthew''<br />
** Miniprepped pBIB<br />
** Ran Digestions and ethanol precipitation<br />
** Ligated with hexameric linkers<br />
** Transformed, had some colonies<br />
<br />
'''Week of June 13-19:''' <br />
<br />
* ''Christian''<br />
** EcoR1 digest of ligation product<br />
** Transformed Top 10 Cells with pBIB from ligation and plated<br />
** Inoculated 30 colonies<br />
** UNR mini prep on all 30 colonies<br />
** Digested Samples with HindIII and EcoR1<br />
** QIAGEN mini prep on samples 11,12 &13<br />
** Digested Samples with HindIII and EcoR1<br />
<br />
* ''Bryson''<br />
** Prepared liquid cultures of pBIB# 1 and pBIB# 2<br />
** Miniprepped liquid cultures of pBIB#<br />
** EcoRI and HinDIII digests of pBIB#<br />
** Ran agarose gel of pBIB cut and uncut, pBIB# uncut, pBIB# cut with EcoRI and pBIB# cut with HinDIII to confirm the absence of EcoRI site in pBIB#<br />
** Single-colony streaked pBIB# 2 on a fresh LB-Kan plate<br />
<br />
* ''Elaine''<br />
** Worked with Chris to incubate 30 liquid cell cultures<br />
** Ran 0.8% gels of all 30 samples<br />
<br />
* ''Randy and Vadim''<br />
** Topocloned ccdB gene into TOPO PCR Blunt II vector<br />
** Determined concentration of pBIB maxipreps using PicoGreen analysis<br />
** Single-colony isolated nine colonies of TOPO-cloned ccdB gene<br />
** Miniprepped cultures of ccdB gene in TOPO-clone<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9<br />
** Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates and digested them<br />
** Ran on gel, some had strange bands, I want to retest<br />
** Colonies appear to not have eliminated EcoRI<br />
<br />
'''Week of June 20-26:'''<br />
<br />
* ''Christian''<br />
** Ran gel of Sample 11<br />
<br />
* ''Bryson''<br />
** Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate<br />
** Started 20 mL liquid cultures of 2-1, 2-2, and 2-3<br />
** Miniprepped samples<br />
** EcoRI and XbaI digests of samples to confirm that the EcoRI was gone and 2-1, 2-2, 2-3 arose from the same colony<br />
** 0.8% agarose gel of digests<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit<br />
** Nanodropped minipreps<br />
** Digested minipreps using EcoRI<br />
** Ran 1% gel for digested minipreps<br />
** Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
** Analyzed ccdB samples using Vector NTI (Invitrogen)<br />
** Sent cell lines 7-2, 8-2, and 9-1 for sequencing/resequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added<br />
<br />
* ''Elaine''<br />
** Primer Design of RD29A<br />
<br />
* ''Matthew''<br />
** Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI<br />
** Team reevaluated its standing, given a month of failed attempts to modify pBIB<br />
** We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak<br />
** I design primers to extract parts from iGEM vectors with Kozak sequences<br />
<br />
==JULY==<br />
'''Week of June 27-July 3:'''<br />
<br />
* ''Randy and Vadim''<br />
** Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit<br />
** Prepared thermal cycler protocol for pBIB vector<br />
** Performed multiple PCR on pBIB vector<br />
** Transformed ccdB into NEB cells unsuccessfully<br />
** Made chlorophenocol resistant agar plates<br />
** Agarose gel analyzed PCR products<br />
<br />
* ''Matthew''<br />
** Transformed colonies with iGEM parts, miniprepped.<br />
** Attempted PCR on EYFP, Failed. Reevaluated protocol.<br />
<br />
'''Week of July 4-10:'''<br />
<br />
* ''Randy and Vadim''<br />
** Agarose gel analyzed PCR products<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made Kanamycin resistant LB plates<br />
<br />
* ''Elaine''<br />
** Transformed ccdB into NEB, OmniMax, and DEB cells<br />
** Made LB/KAN plates<br />
** Took picture of ccdB Sensitivity Experiment 1 Results<br />
** GFP Primer Design<br />
<br />
'''Week of July 11-17:'''<br />
<br />
* ''Elaine'' <br />
** Made LB/Amp plates<br />
<br />
* ''Randy and Vadim''<br />
** TOPOcloned pBIB fragment<br />
** Attempted to ligate ccdB into iGEM vector pSB1C3<br />
** Single colony streaked ccdB transformation<br />
** Made Chloramphenicol broth<br />
** Performed PCR on pBIB cell lines<br />
** Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI<br />
** Miniprepped ccdB colonies<br />
** Gel analyzed pBIB PCR product<br />
** Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates<br />
** Made 50x TAE buffer<br />
** Miniprepped pSB1C3+RFP vector<br />
<br />
* ''Matthew''<br />
** Processed Miniprepped iGEM colonies for mCherry and EYFP<br />
** Set up PCR for mCherry, EYFP, and NOS<br />
<br />
'''Week of July 18-24:'''<br />
<br />
* ''Elaine''<br />
** PCR of GFP<br />
** Ran agarose gel analysis of GFP PCR product<br />
<br />
* ''Randy and Vadim''<br />
** Amplified pBIB fragment using PCR<br />
** Gel analyzed pBIB PCR product<br />
** Attempted ligation of ccdB into iGEM vector pSB1C3<br />
** Transformed ligation into NEB cells<br />
** Selected supposed colonies with ccdB+pSB1C3<br />
<br />
* ''Matthew''<br />
** Analyzed PCR fragments on gels for mCherry, EYFP, and NOS<br />
[[Image: Polasko_eyfpcherry_pcrreal.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
PCR results: worked for eyfp and mcherry with bands around 700bp. <br />
<p>From Left: Lane 1 - Lamda Hind III Mlc. Marker.</p><br />
<p>Lane 2 - Uncut iGem vector</p><br />
<p>Lane 3 - EYFP</p><br />
<p>Lane 4 - EYFP</p><br />
<p>Lane 5 - EYFP</p><br />
<p>Lane 6 - mCherry</p><br />
<p>Lane 7 - mCherry</p><br />
<p>Lane 8 - mCherry</p><br />
<p>Lane 9 - 100bp ladder</p><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** NOS PCR did not appear to work.<br />
** Performed transformation protocol with TOPO vector<br />
** Performed screening on Kan and miniprepped<br />
<br />
'''Week of July 25-31:'''<br />
<br />
* ''Christian''<br />
** Transformed Top 10 cells with pBIB and pMA (containing rd29A)<br />
<br />
* ''Elaine''<br />
** GFP Transformation<br />
** Cell Culture of GFP <br />
** Miniprep of GFP<br />
** EcoR I & Pst Digest of GFP <br />
** Ran 1.2% agarose gel analysis of the GFP digest <br />
** PCR of GFP<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB+pSB1C3 colonies<br />
** Digested minipreps with EcoRI and PstI<br />
** Performed colony PCR on pSB1C3 colonies<br />
<br />
* ''Matthew''<br />
** Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO<br />
** 1 possible candidate each for mChery, EYFP, and NOS.<br />
*** Nos bands did not show but sent one for sequencing anyway<br />
<br />
==AUGUST==<br />
'''Week of August 1-7:'''<br />
<br />
* ''Christian''<br />
** Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region<br />
** Mini prep on pBIB and pMA inoculations<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP PCR product<br />
** Topocloned GFP PCR product <br />
** Cell Culture of GFP Topocloned colonies<br />
** Streaked colony plate<br />
** Miniprep of GFP Topocloned colonies<br />
** EcoR I digestion of GFP Topocloned <br />
** Ligation Test: GFP original vector digested with EcoR I <br />
<br />
* ''Samantha and Christian''<br />
** pMA and pBIB liquid cultures and mini preps<br />
<br />
* ''Bryson''<br />
** Designed and ordered primers for the 35S promoter<br />
** Made spectymycin plates<br />
** Transformed DB3.1 cells with pK7FWG2<br />
** Liquid cultures and minipreps to isolate pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Grew ccdB 8-2 on Kan. resistant plates<br />
** Gel analyzed colony PCR product from July 29<br />
** Made additional LB broth<br />
** Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Performed PCR cleanup on ccdB<br />
<br />
'''Week of August 8-14:'''<br />
<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest<br />
** Ligation Test: Ligated EcoR I digest GFP original vector<br />
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells <br />
** Cell culture and streaked colonies of RFP in PSB1C3 vector <br />
** Miniprepped RFP in PSB1C3 vector<br />
** Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI<br />
** Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors<br />
** Ligated the RFP & GFP in either PSB1C3 & Topo Vectors<br />
** Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates<br />
<br />
* ''Samantha and Christian''<br />
** Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI<br />
** Ligation of triple digest products<br />
<br />
* ''Bryson''<br />
** PCR of pK7FWG2<br />
<br />
* ''Randy and Vadim''<br />
** Made and aliquoted ligase buffer, ATP, and DTT for use for ligation<br />
** Ligated ccdB+pSB1C3, did not use EtOH precipitation<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
** Digested and gel analyzed ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
<br />
'''Week of August 15-21:'''<br />
<br />
* ''Elaine''<br />
** Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates<br />
** Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors<br />
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI<br />
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI<br />
<br />
* ''Bryson''<br />
** 1.2% gel to confirm presence of amplicon<br />
** Topocloning of amplicon and transformation of Topo vector into DB3.1 cells<br />
<br />
*''Randy and Vadim''<br />
** Miniprepped ligation from Aug. 13<br />
** Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
** Autoclaved labware<br />
<br />
'''Week of August 22-28:'''<br />
<br />
* ''Christian''<br />
** Ran PCR on genomic <i>Arabidopsis thaliana</i> DNA using custom primers<br />
** Ran gel of PCR product, no bands corresponding to DREB1C<br />
<br />
* ''Elaine''<br />
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)<br />
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17<br />
** Miniprepped 8 cell cultures and nanodrop<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
<br />
* ''Bryson''<br />
** Isolated and streaked 6 colonies<br />
** Liquid cultures and minipreps of samples<br />
** Digest of samples with EcoRI and PstI to confirm presence of insert<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped colonies from ligation from Aug. 20<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation<br />
*** Ligation was unsuccessful<br />
** Ligated ccdB+pSB1C3 using EtOH precipitation<br />
** Made Chlor. resistant plates<br />
<br />
* ''Richard and Nick''<br />
**Designed BioBrick-compatible primers to amplify CD2+-Promoter ''AtMRP3'' from ''A. thaliana'' genomic DNA<br />
<br />
==SEPTEMBER==<br />
'''Week of August 29-September 4:'''<br />
* ''Elaine''<br />
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)<br />
** Sequencing analysis of colony #11 & #17<br />
<br />
* ''Bryson''<br />
** Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation<br />
** Miniprepped cultures and eluted in 300 microliters<br />
** 50 microliter digests done<br />
*** 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids<br />
*** Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27<br />
*** Ligation was unsuccessful<br />
** Performed PCR on ccdB<br />
** Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation<br />
<br />
* ''Matthew''<br />
** Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.<br />
** Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo<br />
** Screened colonies on Kan and Amp, selected several to miniprep<br />
** Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP<br />
<br />
'''Week of September 5-11:'''<br />
* ''Elaine''<br />
** Sequenced 4 tubes of #17 of GFP in PSB1C3 <br />
** Digested RD29A with Hind III & MfeI-HF<br />
** Analysis of sequence of GFP (PSB1C3)<br />
<br />
* ''Bryson''<br />
** Transformation of ligation reaction<br />
** Plated on chloramphenicol<br />
** Selected four white colonies and single-colony streaked them<br />
** 4 mL liquid cultures of colonies<br />
** Miniprepped samples and eluted in 50 microliters<br />
** 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3 <br />
<br />
* ''Randy and Vadim''<br />
** Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells<br />
*** Transformation unsuccessful<br />
** Made Chlor. resistant plates<br />
** Made new 3M NaOH stock<br />
<br />
* ''Matthew''<br />
** Miniprepped candidates for mCherry and EYFP in Topo vector<br />
** Ran digests and gels on topo candidates and submitted them for sequencing<br />
** Sequencing Results came back positive for mCherry and EYFP in Topo<br />
*** mCherry showed a point mutation but it should not have an effect on translation<br />
<br />
* ''Richard and Nick''<br />
** Re-amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen on the gel; could be due to high sequence homology in AtMRP family<br />
**Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter<br />
**Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers<br />
<br />
'''Week of September 12-18:'''<br />
<br />
* ''Christian''<br />
** PCR of DREB1C with custom primers using extaq instead of platinum taq using <i>Arabidopsis thaliana</i> columbian<br />
** Ran gel of PCR product<br />
<p>[[Image: DREB1C_gel_1.jpg]]</p><br />
* Bands showed at ~500 bp for all samples<br />
** Sample 2 selected for Topo cloning<br />
<br />
* ''Elaine''<br />
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest<br />
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3) <br />
** Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)<br />
<br />
* ''Randy and Vadim''<br />
** Miniprepped ccdB with Mfe site in TOPO vector<br />
** Miniprepped RFP in pSB1C3<br />
** Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst<br />
** Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst<br />
** Gel purified ccdB and pSB1C3 fragments from digested samples<br />
<br />
* Samantha<br />
** Gel extraction of RD29A <br />
<br />
* ''Matthew''<br />
** Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak<br />
** Several candidates possible for sequencing for EYFP and 3 candidates for mCherry<br />
[[Image: Polasko_EYFPigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
EYFP Results - although it's hard to gel w/o mlc. marker. The bands are the right size around 700bp and iGEM pSB1c3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut EYFP candidate 1</p><br />
<p>Lane 3 - EYFP candidate 1</p><br />
<p>Lane 4 - Uncut EYFP candidate 2</p><br />
<p>Lane 5 - EYFP candidate 2</p><br />
<p>Lane 6 - Uncut EYFP candidate 3</p><br />
<p>Lane 7 - EYFP candidate 3</p><br />
<p>Lane 8 - Uncut EYFP candidate 4</p><br />
<p>Lane 9 - EYFP candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
[[Image: Polasko_mCherryigem.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
mCherry Results - although it's hard to gel w/o mlc. marker. The band in Lane 5 the right size around 700bp with the iGEM pSB1C3 backbone at 2kb<br />
<p>From Left: Lane 1 - 100 bp ladder. Did not show</p><br />
<p>Lane 2 - Uncut mCherry candidate 1</p><br />
<p>Lane 3 - mCherry candidate 1</p><br />
<p>Lane 4 - Uncut mCherry candidate 2</p><br />
<p>Lane 5 - mCherry candidate 2</p><br />
<p>Lane 6 - Uncut mCherry candidate 3</p><br />
<p>Lane 7 - mCherry candidate 3</p><br />
<p>Lane 8 - Uncut mCherry candidate 4</p><br />
<p>Lane 9 - mCherry candidate 4</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<br />
'''Week of September 19-25:'''<br />
* ''Elaine''<br />
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry <br />
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.<br />
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures<br />
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I<br />
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel <br />
** Submitted [RD29A + RFP (PSB1C3)] for sequencing <br />
<br />
* ''Randy and Vadim''<br />
** Concentrated gel purification sample from Sept. 16<br />
** Ligated ccdB gene into pSB1C3 vector<br />
** Transformed ccdB+pSB1C3 ligation into DB3 cells<br />
** Miniprepped ccdB+pSB1C3 ligation<br />
<br />
* Samantha<br />
** PCR RD29A from pMA plasmid<br />
** Confirm PCR reaction via agarose gel<br />
<br />
* ''Matthew''<br />
** Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak<br />
** Sequences came back positive for EYFP and mCherry <br />
*** Point mutation in the mCherry but shouldn't affect the translation<br />
<br />
==OCTOBER==<br />
'''Week of September 26-October 2:'''<br />
<br />
* ''Christian''<br />
** Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
<br />
* ''Elaine''<br />
** Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission<br />
<br />
* ''Randy and Vadim''<br />
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst<br />
*** Digest yielded successful ligation<br />
** Made Kan and Amp resistant plates<br />
** Made additional LB broth<br />
** Autoclaved labware and equipment<br />
** Miniprepped ligations from Sept. 23<br />
** Submitted ligation for sequencing at the Nevada Genomics Center<br />
** Made glycerol stock of ligations from Sept. 23<br />
** Transformed ligations from Sept. 23 into NEB cells<br />
*** Plates yielded no growth-ccdB ligation successful<br />
<br />
* Samantha<br />
** Gel purification of RD29A PCR product<br />
<br />
* ''Matthew''<br />
** Submitted Kozak-mCherry and Kozak-EYFP to iGEM<br />
** Cultured and miniprepped some of Elaine's GFP iGEM vector<br />
** Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo<br />
<br />
'''Week of October 3-9:'''<br />
<br />
* ''Christian''<br />
** Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies<br />
** Digested results with EcoR1 and Pst1<br />
** Ran products on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_2.jpg]]</p><br />
* Expected bands show at ~550 bp<br />
** Sent samples for sequencing analysis<br />
** Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1<br />
** EtOH ppt of completed digest<br />
** T4 ligation (using custom buffer)<br />
** Digested Topocloned DREB1C_2 with EcoR1 and Spe1<br />
** Digested YFP_pSB1C3 with EcoR1 and Xba1<br />
** EtOH ppt of DREB1C/YFP digest products<br />
** T4 ligation (using custom buffer)<br />
** Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection<br />
<br />
* ''Elaine''<br />
** Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.<br />
<p>[[Image:Rd29A PCR product - Samantha Lee.jpg|500px]]</p><br />
* The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp. <br />
** Topocloned Samantha Lee's PCR product #1 of rd29A<br />
** Streaked single colonies of rd29A (Topo Vector)<br />
** Cell cultured single colonies of rd29A (Topo Vector)<br />
** Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1. <br />
<br />
* ''Matthew''<br />
** Completed ligation and began transformation for 35S-GFP candidates<br />
** Screened 35S-GFP candidates on chloramphenicol and kanamycin <br />
** Cultured,miniprepped,digested, and ran on gel 20 candidates<br />
*** One candidate possible for sequencing<br />
[[Image: Polasko_35sgfp.jpg|300px|thumb|left]]<br />
<p>&nbsp;</p><br />
35S-GFP Composite Results - With careful inspection of my gels, I believed Lane 3 was a good candidate. I was looking for 2 bands, the composite at about 1.8kb and the pSB1C3 backbone at 2kb. Although, the image is not clean, I believed I saw a second band emerging in between the molecular markers which were 2kb and 1.6kb, making that emerging band at 1.8kb. Sequencing would prove me right<br />
<p>From Left: Lane 1 - 1 kb ladder.</p><br />
<p>Lane 2 - Uncut 35S-GFP candidate 1</p><br />
<p>Lane 3 - 35S-GFP candidate 1</p><br />
<p>Lane 4 - Uncut 35S-GFP candidate 2</p><br />
<p>Lane 5 - 35S-GFP candidate 2</p><br />
<p>Lane 6 - Uncut 35S-GFP candidate 3</p><br />
<p>Lane 7 - 35S-GFP candidate 3</p><br />
<p>Lane 8 - Uncut 35S-GFP candidate 4</p><br />
<p>Lane 9 - 35S-GFP candidate 4</p><br />
<p>Lane 10 - 35S-GFP candidate 5</p><br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
** Submitted for sequencing 1 35S-GFP candidate<br />
<br />
'''Week of October 10-16:'''<br />
<br />
<br />
* ''Elaine''<br />
** Ran rd29A (Topo Vector) gel.<br />
<p>[[Image: Rd29A in TOPO Vector.jpg|500px]]</p><br />
* The "rd29A in Topo Vector" gel analysis was successful for it showed the rd29A bands at ~836 bp and the Topo bands at ~3,519 bp. (Digested/Cut samples with EcoR1 and Pst 1 = labeled C5, C6, C7, & C8).<br />
** Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.<br />
** Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).<br />
** Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).<br />
<br />
<br />
* ''Christian''<br />
** Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection<br />
** 5 colonies selected and inoculated overnight at 37C<br />
** Miniprep 5 cultures and digested with EcoR1 and Pst1<br />
** Ran digests on 0.8% agarose gel<br />
<p>[[Image: DREB1C_gel_3.jpg]]</p><br />
* Expected bands appeared at ~500 bp for sample 3 and sample 5<br />
** Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing<br />
** Sequencing data for DREB1C_2.3 had 0 mismatches and was selected<br />
<br />
* ''Matthew''<br />
** Sequencing results came back positive for 35S-GFP<br />
** Miniprepped 35S-GFP and made glycerol stock<br />
<br />
* ''Richard and Nick''<br />
** Amplified CD2+ promoter from A. thaliana genomic DNA with ExTaq polymerase; several unspecific bands were seen again on the gel; performed gel extraction on fragments of interest<br />
<br />
<br />
<br />
<br />
'''Week of October 17-23:'''<br />
<br />
* ''Elaine''<br />
** 30 Cell cultures of rd29A (pSB1C3)<br />
** Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested. <br />
** Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector<br />
** These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"<br />
<p>[[Image:Rd29A in pSB1C3 - Epic Failure.jpg|500px]]</p><br />
* The "rd29A in pSB1C3 iGEM Vector" gel analysis was NOT successful for the expected bands of the rd29A at ~836bp didn't show up on the digested (cut with EcoR1 & Pst 1) samples labeled C1, C3, C13, & C15.<br />
* Due to the 30 single colonies that was unsuccessful in having the rd29A in pSB1C3 iGEM Vector, I proceeded to test 45 single colonies by using the colony PCR Technique. <br />
** Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels<br />
<p>[[Image:Colony PCR -21 - rd29A in pSB1C3 Vector.jpg|500px]]</p><br />
* The "Colony PCR # 21 of rd29A in pSB1C3 iGEM Vector" gel analysis was successful for 1 (colony PCR #21) out of 45 colonies showed an rd29A band at ~836bp.(Band is shown in the Lane labeled #21).<br />
** Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4). <br />
** Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer. <br />
** Sequenced Colony PCR #21 at the Nevada Genomics Center.<br />
** Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.<br />
** Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3. <br />
<br />
* ''Christian''<br />
** Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007<br />
** 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers<br />
** Products of PCR reaction run on a 0.8% agarose gel<br />
[[Image: DREB1C_gel_4.jpg]]<br />
* Band at ~500 bp for colony 24<br />
** Colony 24 was the only positive result<br />
** Colony 24 DREB1C/YFP construct was sent for sequencing<br />
<br />
* ''Matthew''<br />
** Submitted 35S-GFP to iGEM<br />
<br />
* ''Richard and Nick''<br />
** Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board<br />
<br />
<br />
'''Week of October 24-30:'''<br />
<br />
<br />
* ''Elaine''<br />
** Added rd29A (pSB1C3) into the registry<br />
** Submitted rd29A (pSB1C3) through the mail.<br />
<br />
<br />
<p>&nbsp;</p><br />
<p>&nbsp;</p><br />
----<br />
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