Team:Nevada/Notebook

From 2010.igem.org

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** Cell Culture of GFP  
** Cell Culture of GFP  
** Miniprep of GFP
** Miniprep of GFP
-
** EcoR I & Pst Digest of GFP  
+
** EcoR 1 & Pst 1 Digest of GFP  
** Ran 1.2% agarose gel analysis of the GFP digest  
** Ran 1.2% agarose gel analysis of the GFP digest  
 +
<p>[[Image:GFP from iGEM (E0040) paint.jpg|500px]]</p>
 +
* 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).
** PCR of GFP
** PCR of GFP
Line 381: Line 383:
* ''Elaine''
* ''Elaine''
** Ran 1.2% Agarose Gel analysis of GFP PCR product
** Ran 1.2% Agarose Gel analysis of GFP PCR product
 +
<p>[[Image:GFP PCR product.jpg|500px]]</p>
 +
* 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)
** Topocloned GFP PCR product  
** Topocloned GFP PCR product  
** Cell Culture of GFP Topocloned colonies
** Cell Culture of GFP Topocloned colonies
Line 409: Line 413:
* ''Elaine''
* ''Elaine''
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest
** Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest
 +
<p>[[Image:GFP in Topo Vector 001.jpg|500px]]</p>
 +
* 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.
** Ligation Test: Ligated EcoR I digest GFP original vector
** Ligation Test: Ligated EcoR I digest GFP original vector
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells  
** Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells  
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** Digested GFP in PSB1C3 vectors with EcoR1 & PstI
** Digested GFP in PSB1C3 vectors with EcoR1 & PstI
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI
** Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI
 +
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p>
 +
* 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.
* ''Bryson''
* ''Bryson''
Line 461: Line 469:
* ''Elaine''
* ''Elaine''
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)
** Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)
-
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony # 17
+
** Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony #17
** Miniprepped 8 cell cultures and nanodrop
** Miniprepped 8 cell cultures and nanodrop
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
Line 484: Line 492:
* ''Elaine''
* ''Elaine''
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
** Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
 +
** Analysis of sequence of GFP (PSB1C3)
** Sequencing analysis of colony #11 & #17
** Sequencing analysis of colony #11 & #17
Line 508: Line 517:
* ''Elaine''
* ''Elaine''
** Sequenced 4 tubes of #17 of GFP in PSB1C3  
** Sequenced 4 tubes of #17 of GFP in PSB1C3  
-
** Digested RD29A with Hind III & MfeI-HF (for Chris' Project)
+
** Digested rd29A with Hind III & MfeI-HF (for Chris' Project)
-
** Analysis of sequence of GFP (PSB1C3)
+
-
<p>[[Image:GFP in pSB1C3 iGEM Vector.jpg|500px]]</p>
+
-
* 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.
+
* ''Bryson''
* ''Bryson''
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* ''Elaine''
* ''Elaine''
-
** RD29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest
+
** rd29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)  
** Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)  
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)  
** Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)  
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** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry  
** 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry  
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.
** 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.
-
** Streaked single colonies [RD29A + RFP (PSB1C3)] and did Cell cultures
+
** Streaked single colonies [rd29A + RFP (PSB1C3)] and did Cell cultures
-
** Miniprepped [RD29A + RFP (PSB1C3)]and digested with EcoR I and Pst I
+
** Miniprepped [rd29A + RFP (PSB1C3)]and digested with EcoR I and Pst I
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel  
** Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel  
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p>
<p>[[Image:Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg|500px]]</p>
* 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).
* 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).
-
** Submitted [RD29A + RFP (PSB1C3)] for sequencing  
+
** Submitted [rd29A + RFP (PSB1C3)] for sequencing  
* ''Randy and Vadim''
* ''Randy and Vadim''
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** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst
** Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst
*** Digest yielded successful ligation
*** Digest yielded successful ligation
 +
http://2010.igem.org/wiki/images/b/bf/CcdB%2BpSB1C3_gel.png
** Made Kan and Amp resistant plates
** Made Kan and Amp resistant plates
** Made additional LB broth
** Made additional LB broth
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!align="center"|[[Image:Promega_logo.jpg‎]]
!align="center"|[[Image:Promega_logo.jpg‎]]
!align="center"|[[Image:Invitrogen_logo.jpeg]]
!align="center"|[[Image:Invitrogen_logo.jpeg]]
-
 
+
!align="center"|[[Image:Sda logo small.jpg]]
|}
|}

Latest revision as of 21:20, 27 October 2010

UNR Notebook.png

 

 

Team Nevada Notebook


Contents

APRIL

Week of April 11-17:

  • Christian
    • Transformed pBIB into Top 10 Cells
  • Bryson, Michael, Senny, Tyler
    • Made tobacco cell (NT-1) media in Dr. Shintani's lab

Week of April 18-24:

  • Bryson, Christian
    • EcoRI digest of pBIB
    • Made Na acetate buffer
  • Christian
    • Ran gel of EcoR1 Digested pBIB

MAY

Week of April 25-May 1:

  • Bryson
    • Ran agarose gel of EcoRI digest
  • Matthew
    • Team collaborated on pBIB assignment. Too many chefs in the kitchen.
    • We decide to each tackle the pBIB problem in parallel

Week of May 2-8:

  • Elaine
    • Ran 0.8% agarose gel of EcoRI digest
    • Made LB/KAN plates
    • Spread for colonies
    • Did miniprep for pBIB liquid cultures
  • Matthew
    • Miniprepped pBIB
    • Ran EcoRI digest and Klenow
    • Ethanol precipitated and ligated

Week of May 9-15:

  • Christian
    • EcoR1/Xba1 Digest of pBIB
    • Did Mini prep on 4 cultures
  • Bryson
    • Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.
    • Did minipreps on additional pBIB liquid cultures.
  • Elaine
    • Did XbaI and EcoRI digest of pBIB
    • Ran 2 0.8% gels of each digest
    • Did a miniprep and a Phenol:chloroform clean-up
    • Ran 0.8% gel of the XbaI and EcoRI digest with the uncut pBIB
  • Matthew
    • Ligated and transformed
    • Screened colonies, miniprepped, and digested, ran on gel
    • No candidates had EcoRI eliminated

Week of May 16-22:

  • Christian
    • Generated glycerol stock of pBIB transformed Top 10 cells
    • Inoculated 5 cultures of pBIB transformed Top 10 cells grown on Kan resistant plates
    • Ran samples on gel after digest with EcoR1
  • Bryson
    • Klenow reactions of EcoRI digests
    • Phenol:chloroform cleanup of pBIB prior to ligation
    • Blunt-end ligation of klenowed pBIB
  • Randy Pares and Vidim Gladwell
    • Designed primers pBIB-RB-F, pBIB-RB-R, NOS 3'-Foward Nos3' reverse for sequencing the pBIB plant plasmid.
  • Elaine
    • Made LB/KAN plates
    • Made 50mg/ml stock of KAN
    • Made 1X TAE buffer
  • Matthew
    • Digested and tested more colonies, none worked
    • Started over with pBIB and Klenow
    • Ethanol precipitated and ligated
    • Transformed
    • Screened colonies, miniprepped, and digested, ran on gel
    • No candidates had EcoRI eliminated

Week of May 23-29:

  • Christian
    • Miniprep on pBIB transformed Top 10 cells using "low copy #" modification
    • Digested with EcoR1 and HindIII
  • Bryson
    • Incubated 50 mL liquid culture of E. coli with pBIB (samples 3).
    • Miniprepped sample 3, using modified protocol for large/low-copy plasmids.
    • EcoRI digest of uncut sample 3
    • Prepared 5 mM dNTP stock
  • Elaine
    • Incubated 50 mL liquid culture of E. coli with pBIB (sample 4 & sample 5)
    • Qiaprep miniprep of sample 4 & sample 5 according to manufacturer's modified protocol for large/low-copy plasmids
    • Nanodrop of DNA recovery of miniprepped sample 4 & sample 5
  • Randy and Vadim
    • Sent pBIB vector to Nevada Genomics Center for sequencing with primers designed during the week of May 16-22
    • Designed forward and reverse minimal ccdB primers for PCR and sequencing of ccdB gene
    • Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit
  • Matthew
    • Investigated alternative ways of eliminating EcoRI site.
    • Want to try hexameric linkers

JUNE

Week of May 30-June 5:

  • Christian
    • Miniprep Top 10 Cells transformed with pBIB
  • Bryson
    • HinD3 digest of uncut sample 3
    • Ran 0.8% gels of samples 1-5 to verify complete digestion by EcoRI
    • Pooled uncut samples 3, 4 and 5 (pBIB-pool)
    • Recieved 5 µg of pBIB from Randy and Vadim's maxi-prep (pBIB-maxi)
    • EcoRI digest of pBIB-pool and pBIB-maxi
    • Ran 0.8% agarose gel of EcoRI digests
    • Klenow reactions of pBIB-pool and pBIB-maxi
    • Made glycerol stocks of pBIB samples 1-5
  • Elaine
    • EcoRI digest of uncut pBIB sample 4 and 5
    • HinD3 digest of uncut sample 4 & sample 5 as a positive control
    • Ran 0.8% gels of samples 1-5 to see if EcoRI digest was successful
    • Ethanol precipitation of the EcoRI digests of sample 4 & sample 5
    • Nanodrop resulted to a low DNA content
    • Worked with Bryson for the EcoRI digest of the 5µg of pBIB maxi-prep
    • Ran 0.8% agarose gel of the pBIB maxi-prep EcoRI digest
    • Modified all protocols of the Binary vector
  • Randy and Vadim
    • Calculated amount of reagent needed for Deep Vent DNA polymerase reaction (New England Biolabs)
    • Programmed thermal cycler for PCR of ccdB gene
    • Ran PCR for ccdB
    • Prepared LB/KAN Broth
    • Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel
      • Reaction was unsuccessful
  • Matthew
    • Miniprepped pBIB
    • Ran EcoRI digests
    • Ran ethanol precipitation
    • Ligated with several concentrations of hexameric linkers designed to destroy site
    • Ran transformation, no colonies
      • Believed procedures/conditions ran on ligation and transformation not ideal

Week of June 6-12:

  • Christian
    • Klenow reaction on EcoR1 digested pBIB
    • T4 ligation on Klenow products
  • Bryson
    • Ligation reactions for pBIB-pool and pBIB-maxi
    • Digested pBIB-pool and pBIB-maxi again with EcoRI to linearize any unmodified pBIB
    • Transformed Top-10 Cells with modified pBIB (designated pBIB#)
      • Obtained two colonies after overnight incubation
    • Line-streaked the two colonies (pBIB# 1 and pBIB# 2) on an LB-Kan plate
  • Randy and Vadim
    • Uploaded pBI101, pBIN19, pBIB-KAN, and ccdB gene to Vector NTI
    • Ordered second set of pBIB primers: pBIB-RB-F2, pBIB-RB-F3, pBIB-RB-R2, and pBIB-RB-R3 for sequencing of pBIB
    • Modified thermal cycler conditions for PCR of ccdB gene
    • Ran PCR for ccdB using HiFi DNA polymerase (Invitrogen)
    • Gel analyzed resultant ccdB PCR reaction with 1.2% agarose gel and successfully amplified ccdB gene

PCR_ccdB_confirmation.png

    • Maxiprepped pBIB vector using QIAGEN QIAfilter Plasmid Maxi Kit
  • Matthew
    • Miniprepped pBIB
    • Ran Digestions and ethanol precipitation
    • Ligated with hexameric linkers
    • Transformed, had some colonies

Week of June 13-19:

  • Christian
    • EcoR1 digest of ligation product
    • Transformed Top 10 Cells with pBIB from ligation and plated
    • Inoculated 30 colonies
    • UNR mini prep on all 30 colonies
    • Digested Samples with HindIII and EcoR1
    • QIAGEN mini prep on samples 11,12 &13
    • Digested Samples with HindIII and EcoR1
  • Bryson
    • Prepared liquid cultures of pBIB# 1 and pBIB# 2
    • Miniprepped liquid cultures of pBIB#
    • EcoRI and HinDIII digests of pBIB#
    • 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#
    • Single-colony streaked pBIB# 2 on a fresh LB-Kan plate
  • Elaine
    • Worked with Chris to incubate 30 liquid cell cultures
    • Ran 0.8% gels of all 30 samples
  • Randy and Vadim
    • Topocloned ccdB gene into TOPO PCR Blunt II vector
    • Determined concentration of pBIB maxipreps using PicoGreen analysis
    • Single-colony isolated nine colonies of TOPO-cloned ccdB gene
    • Miniprepped cultures of ccdB gene in TOPO-clone
    • Digested minipreps using EcoRI
    • Ran 1% gel for digested minipreps
    • Single colony streaked four cell lines of ccdB gene in TOPO-clone: line 3, line 7, line 8, line 9
    • Ordered primers for Left Border Repeat of pBIB: pBIB-LB-F and pBIB-LB-R for sequencing of pBIB vector
  • Matthew
    • Miniprepped candidates and digested them
    • Ran on gel, some had strange bands, I want to retest
    • Colonies appear to not have eliminated EcoRI

Week of June 20-26:

  • Christian
    • Ran gel of Sample 11
  • Bryson
    • Selected 3 colonies from pBIB# 2 (2-1, 2-2, 2-3) and streaked on a fresh LB-Kan plate
    • Started 20 mL liquid cultures of 2-1, 2-2, and 2-3
    • Miniprepped samples
    • 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
    • 0.8% agarose gel of digests
  • Randy and Vadim
    • Miniprepped isolated cultures of ccdB gene using QIAGEN QIAprep Spin Miniprep Kit
    • Nanodropped minipreps
    • Digested minipreps using EcoRI
    • Ran 1% gel for digested minipreps
    • Sent cell lines 7-2 and 8-2 for sequencing to Nevada Genomics Center with ccdB M13 Forward and Reverse primers added
    • Analyzed ccdB samples using Vector NTI (Invitrogen)
    • 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
  • Elaine
    • Primer Design of RD29A
  • Matthew
    • Attempted to test more candidates from hexameric ligation, no candidates had eliminated EcoRI
    • Team reevaluated its standing, given a month of failed attempts to modify pBIB
    • We are each assigned new tasks. I will be put in charge of making EYFP and mCherry with plant Kozak
    • I design primers to extract parts from iGEM vectors with Kozak sequences

JULY

Week of June 27-July 3:

  • Randy and Vadim
    • Prepared ccdB for MaxiPrep using QIAGEN QIAfilter Plasmid Maxi Kit
    • Prepared thermal cycler protocol for pBIB vector
    • Performed multiple PCR on pBIB vector
    • Transformed ccdB into NEB cells unsuccessfully
    • Made chlorophenocol resistant agar plates
    • Agarose gel analyzed PCR products
  • Matthew
    • Transformed colonies with iGEM parts, miniprepped.
    • Attempted PCR on EYFP, Failed. Reevaluated protocol.

Week of July 4-10:

  • Randy and Vadim
    • Agarose gel analyzed PCR products
    • Transformed ccdB into NEB, OmniMax, and DEB cells
    • Made Kanamycin resistant LB plates
  • Elaine
    • Transformed ccdB into NEB, OmniMax, and DEB cells
    • Made LB/KAN plates
    • Took picture of ccdB Sensitivity Experiment 1 Results
    • GFP Primer Design

Week of July 11-17:

  • Elaine
    • Made LB/Amp plates
  • Randy and Vadim
    • TOPOcloned pBIB fragment
    • Attempted to ligate ccdB into iGEM vector pSB1C3
    • Single colony streaked ccdB transformation
    • Made Chloramphenicol broth
    • Performed PCR on pBIB cell lines
    • Digested ccdB in iGEM vector pSB1C3 with PstI and EcoRI
    • Miniprepped ccdB colonies
    • Gel analyzed pBIB PCR product
    • Transformed pSB1C3+RFP vector onto Chloramphenicol resistant plates
    • Made 50x TAE buffer
    • Miniprepped pSB1C3+RFP vector
  • Matthew
    • Processed Miniprepped iGEM colonies for mCherry and EYFP
    • Set up PCR for mCherry, EYFP, and NOS

Week of July 18-24:

  • Elaine
    • PCR of GFP
    • Ran agarose gel analysis of GFP PCR product
  • Randy and Vadim
    • Amplified pBIB fragment using PCR
    • Gel analyzed pBIB PCR product
    • Attempted ligation of ccdB into iGEM vector pSB1C3
    • Transformed ligation into NEB cells
    • Selected supposed colonies with ccdB+pSB1C3
  • Matthew
    • Analyzed PCR fragments on gels for mCherry, EYFP, and NOS
Polasko eyfpcherry pcrreal.jpg

 

 

 

PCR results: worked for eyfp and mcherry with bands around 700bp.

From Left: Lane 1 - Lamda Hind III Mlc. Marker.

Lane 2 - Uncut iGem vector

Lane 3 - EYFP

Lane 4 - EYFP

Lane 5 - EYFP

Lane 6 - mCherry

Lane 7 - mCherry

Lane 8 - mCherry

Lane 9 - 100bp ladder

 

 

 

 

    • NOS PCR did not appear to work.
    • Performed transformation protocol with TOPO vector
    • Performed screening on Kan and miniprepped

Week of July 25-31:

  • Christian
    • Transformed Top 10 cells with pBIB and pMA (containing rd29A)
  • Elaine
    • GFP Transformation
    • Cell Culture of GFP
    • Miniprep of GFP
    • EcoR 1 & Pst 1 Digest of GFP
    • Ran 1.2% agarose gel analysis of the GFP digest

GFP from iGEM (E0040) paint.jpg

  • 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).
    • PCR of GFP
  • Randy and Vadim
    • Miniprepped ccdB+pSB1C3 colonies
    • Digested minipreps with EcoRI and PstI
    • Performed colony PCR on pSB1C3 colonies
  • Matthew
    • Ran Digests and gels for candidates of mCherry, EYFP, and NOS in TOPO
    • 1 possible candidate each for mChery, EYFP, and NOS.
      • Nos bands did not show but sent one for sequencing anyway

AUGUST

Week of August 1-7:

  • Christian
    • Designed and ordered primers for wt Arabidopsis isolation of DREB1C promoter region
    • Mini prep on pBIB and pMA inoculations
  • Elaine
    • Ran 1.2% Agarose Gel analysis of GFP PCR product

GFP PCR product.jpg

  • 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)
    • Topocloned GFP PCR product
    • Cell Culture of GFP Topocloned colonies
    • Streaked colony plate
    • Miniprep of GFP Topocloned colonies
    • EcoR I digestion of GFP Topocloned
    • Ligation Test: GFP original vector digested with EcoR I
  • Samantha and Christian
    • pMA and pBIB liquid cultures and mini preps
  • Bryson
    • Designed and ordered primers for the 35S promoter
    • Made spectymycin plates
    • Transformed DB3.1 cells with pK7FWG2
    • Liquid cultures and minipreps to isolate pK7FWG2
  • Randy and Vadim
    • Grew ccdB 8-2 on Kan. resistant plates
    • Gel analyzed colony PCR product from July 29
    • Made additional LB broth
    • Gel analyzed ccdB+pSB1C3 ligation using EcoRI and Pst
      • Ligation was unsuccessful
    • Performed PCR cleanup on ccdB

Week of August 8-14:

  • Elaine
    • Ran 1.2% Agarose Gel analysis of GFP Topocloned EcoR I digest

GFP in Topo Vector 001.jpg

  • 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.
    • Ligation Test: Ligated EcoR I digest GFP original vector
    • Ligation Test: Transformed Ligation and EcoR I digest of GFP into NEB cells
    • Cell culture and streaked colonies of RFP in PSB1C3 vector
    • Miniprepped RFP in PSB1C3 vector
    • Digested both RFP in PSB1C3 vector & GFP Topo Vector together with EcoRI and PstI
    • Ethanol Precipitation of Digested RFP & GFP in PSB1C3 & Topo Vectors
    • Ligated the RFP & GFP in either PSB1C3 & Topo Vectors
    • Transformed RFP & GFP in either PSB1C3 & Topo Vectors by growing in NEB Cells in KAN & Chloramphenicol plates
  • Samantha and Christian
    • Triple digest of pMA and pBIB with Hind III, Mfe I, and Eco RI
    • Ligation of triple digest products
  • Bryson
    • PCR of pK7FWG2
  • Randy and Vadim
    • Made and aliquoted ligase buffer, ATP, and DTT for use for ligation
    • Ligated ccdB+pSB1C3, did not use EtOH precipitation
    • Miniprepped ccdB+pSB1C3 ligation
    • Digested and gel analyzed ligation using EcoRI and Pst
      • Ligation was unsuccessful
    • Ligated ccdB+pSB1C3 using EtOH precipitation

Week of August 15-21:

  • Elaine
    • Selected 20 colonies for cell culture and streaked into KAN & Chloramphenicol plates
    • Miniprepped 11 cell cultures that didn't grow in KAN plates which means it has to be GFP in PSB1C3 vectors
    • Digested GFP in PSB1C3 vectors with EcoR1 & PstI
    • Ran 1.2% Agarose Gels analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI

GFP in pSB1C3 iGEM Vector.jpg

  • 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.
  • Bryson
    • 1.2% gel to confirm presence of amplicon
    • Topocloning of amplicon and transformation of Topo vector into DB3.1 cells
  • Randy and Vadim
    • Miniprepped ligation from Aug. 13
    • Digested and analyzed ccdB+pSB1C3 ligation using EcoRI and Pst
      • Ligation was unsuccessful
    • Ligated ccdB+pSB1C3 using EtOH precipitation
    • Made Chlor. resistant plates
    • Autoclaved labware

Week of August 22-28:

  • Christian
    • Ran PCR on genomic Arabidopsis thaliana DNA using custom primers
    • Ran gel of PCR product, no bands corresponding to DREB1C
  • Elaine
    • Sequenced colony #11 & #17 of GFP in the PSB1C3 vectors (submitted 4: 11F, 11R, 17F, 17R)
    • Made chloramphenicol plates and cell cultures from colony #17 & streaked more of colony #17
    • Miniprepped 8 cell cultures and nanodrop
    • Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
  • Bryson
    • Isolated and streaked 6 colonies
    • Liquid cultures and minipreps of samples
    • Digest of samples with EcoRI and PstI to confirm presence of insert
  • Randy and Vadim
    • Miniprepped colonies from ligation from Aug. 20
    • Digested and gel-analyzed ccdB+pSB1C3 ligation
      • Ligation was unsuccessful
    • Ligated ccdB+pSB1C3 using EtOH precipitation
    • Made Chlor. resistant plates
  • Richard and Nick
    • Designed BioBrick-compatible primers to amplify CD2+-Promoter AtMRP3 from A. thaliana genomic DNA

SEPTEMBER

Week of August 29-September 4:

  • Elaine
    • Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
    • Analysis of sequence of GFP (PSB1C3)
    • Sequencing analysis of colony #11 & #17
  • Bryson
    • Grew 20 mL liquid cultures of pSB1C3 and Topo vector to prepare for ligation
    • Miniprepped cultures and eluted in 300 microliters
    • 50 microliter digests done
      • 20 microliters were run on 1.0% agarose gel to confirm complete digestion of both plasmids
      • Digested plasmids were added in a roughly 1:1 ratio and ligated in a 40 microliter ligation reaction
  • Randy and Vadim
    • Digested and gel-analyzed ccdB+pSB1C3 ligation from Aug. 27
      • Ligation was unsuccessful
    • Performed PCR on ccdB
    • Ligated ccdB+pSB1C3 using Phenol:Chloroform cleanup techniques and EtOH precipitation
  • Matthew
    • Sequence results from first attempt at mCherry,EYFP, and NOS failed. NOS abandoned.
    • Started over with new colonies, miniprepped, digested, ethanol precipitated, and ligated for Topo
    • Screened colonies on Kan and Amp, selected several to miniprep
    • Digested and ran on gels, identified several more candidates for sequencing for mCherry and EYFP

Week of September 5-11:

  • Elaine
    • Sequenced 4 tubes of #17 of GFP in PSB1C3
    • Digested rd29A with Hind III & MfeI-HF (for Chris' Project)
  • Bryson
    • Transformation of ligation reaction
    • Plated on chloramphenicol
    • Selected four white colonies and single-colony streaked them
    • 4 mL liquid cultures of colonies
    • Miniprepped samples and eluted in 50 microliters
    • 20 microliter digests with EcoRI and PstI done to ensure insert was present in pSB1C3
  • Randy and Vadim
    • Transformed ccdB+pSB1C3 ligation from Sept. 3 into DB3 cells
      • Transformation unsuccessful
    • Made Chlor. resistant plates
    • Made new 3M NaOH stock
  • Matthew
    • Miniprepped candidates for mCherry and EYFP in Topo vector
    • Ran digests and gels on topo candidates and submitted them for sequencing
    • Sequencing Results came back positive for mCherry and EYFP in Topo
      • mCherry showed a point mutation but it should not have an effect on translation
  • Richard and Nick
    • 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
    • Ran PCR product on a long gel and performed a gel extraction to isolate fragments that could be the CD2+ promoter
    • Performed diagnostic digest of fragment extracted; fragment did not yield predicted digestion patterm; designed new primers

Week of September 12-18:

  • Christian
    • PCR of DREB1C with custom primers using extaq instead of platinum taq using Arabidopsis thaliana columbian
    • Ran gel of PCR product

DREB1C gel 1.jpg

  • Bands showed at ~500 bp for all samples
    • Sample 2 selected for Topo cloning
  • Elaine
    • rd29A(pMA) with RFP(PSB1C3) EcoR1 & Pst I Digest
    • Ethanol Precipitation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)
    • Ligation of digested synthetic RD29A + RFP(pMA) with RFP(PSB1C3)
    • Transformation of ligated synthetic RD29A + RFP(pMA) with RFP(PSB1C3)
  • Randy and Vadim
    • Miniprepped ccdB with Mfe site in TOPO vector
    • Miniprepped RFP in pSB1C3
    • Digested and gel analyzed ccdB with Mfe samples using EcoRI and Pst
    • Digested ccdB in TOPO vector and RFP in pSB1C3 using EcoRI and Pst
    • Gel purified ccdB and pSB1C3 fragments from digested samples
  • Samantha
    • Gel extraction of RD29A
  • Matthew
    • Miniprepped, digested, and ran on gels candidates for mCherry and EYFP with Kozak
    • Several candidates possible for sequencing for EYFP and 3 candidates for mCherry
Polasko EYFPigem.jpg

 

 

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

From Left: Lane 1 - 100 bp ladder. Did not show

Lane 2 - Uncut EYFP candidate 1

Lane 3 - EYFP candidate 1

Lane 4 - Uncut EYFP candidate 2

Lane 5 - EYFP candidate 2

Lane 6 - Uncut EYFP candidate 3

Lane 7 - EYFP candidate 3

Lane 8 - Uncut EYFP candidate 4

Lane 9 - EYFP candidate 4

 

 

 

 

Polasko mCherryigem.jpg

 

 

 

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

From Left: Lane 1 - 100 bp ladder. Did not show

Lane 2 - Uncut mCherry candidate 1

Lane 3 - mCherry candidate 1

Lane 4 - Uncut mCherry candidate 2

Lane 5 - mCherry candidate 2

Lane 6 - Uncut mCherry candidate 3

Lane 7 - mCherry candidate 3

Lane 8 - Uncut mCherry candidate 4

Lane 9 - mCherry candidate 4

 

 

 

 

Week of September 19-25:

  • Elaine
    • 09/17/10 Submitted GFP with Kozak sequence (PSB1C3) into registry
    • 09/20/10 Mailed GFP with Kozak sequence (PSB1C3) to MIT.
    • Streaked single colonies [rd29A + RFP (PSB1C3)] and did Cell cultures
    • Miniprepped [rd29A + RFP (PSB1C3)]and digested with EcoR I and Pst I
    • Ran uncut and digested [RD29A + RFP (PSB1C3)] into 1.2 Agarose Gel

Rd29A promoter + RFP in pSB1C3 iGEM Vector.jpg

  • 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).
    • Submitted [rd29A + RFP (PSB1C3)] for sequencing
  • Randy and Vadim
    • Concentrated gel purification sample from Sept. 16
    • Ligated ccdB gene into pSB1C3 vector
    • Transformed ccdB+pSB1C3 ligation into DB3 cells
    • Miniprepped ccdB+pSB1C3 ligation
  • Samantha
    • PCR RD29A from pMA plasmid
    • Confirm PCR reaction via agarose gel
  • Matthew
    • Miniprepped and submitted candidates for sequencing for mCherry and EYFP with Kozak
    • Sequences came back positive for EYFP and mCherry
      • Point mutation in the mCherry but shouldn't affect the translation

OCTOBER

Week of September 26-October 2:

  • Christian
    • Topocloned DREB1C PCR product and spread on plates using Kan/Amp counter selection
    • 5 colonies selected and inoculated overnight at 37C
  • Elaine
    • Miniprepped some more [RD29A + RFP (PSB1C3)] samples for submission
  • Randy and Vadim
    • Digested and gel-analyzed ccdB-pSB1C3 ligation with EcoRI and Pst
      • Digest yielded successful ligation

CcdB%2BpSB1C3_gel.png

    • Made Kan and Amp resistant plates
    • Made additional LB broth
    • Autoclaved labware and equipment
    • Miniprepped ligations from Sept. 23
    • Submitted ligation for sequencing at the Nevada Genomics Center
    • Made glycerol stock of ligations from Sept. 23
    • Transformed ligations from Sept. 23 into NEB cells
      • Plates yielded no growth-ccdB ligation successful
  • Samantha
    • Gel purification of RD29A PCR product
  • Matthew
    • Submitted Kozak-mCherry and Kozak-EYFP to iGEM
    • Cultured and miniprepped some of Elaine's GFP iGEM vector
    • Miniprepped, digested, ethanol precipitated, and began ligation on GFP iGEM with 35S in Topo

Week of October 3-9:

  • Christian
    • Mini prep of 5 (DREB1C_1, DREB1C_2 etc) DREB1C Topo construct colonies
    • Digested results with EcoR1 and Pst1
    • Ran products on 0.8% agarose gel

DREB1C gel 2.jpg

  • Expected bands show at ~550 bp
    • Sent samples for sequencing analysis
    • Digested DREB1C_2 and RFP_pSB1C3 with EcoR1 and Pst1
    • EtOH ppt of completed digest
    • T4 ligation (using custom buffer)
    • Digested Topocloned DREB1C_2 with EcoR1 and Spe1
    • Digested YFP_pSB1C3 with EcoR1 and Xba1
    • EtOH ppt of DREB1C/YFP digest products
    • T4 ligation (using custom buffer)
    • Transformed DREB1C/YFP ligation product into TOP 10 Cells and streaked using Kan/Chlor counterselection
  • Elaine
    • Analyzed rd29A PCR product ran on an Agarose Gel by Samantha Lee.

Rd29A PCR product - Samantha Lee.jpg

  • The "rd29A PCR product" gel analysis was successful for the rd29A PCR products of both #1 & #2 showed bands of the rd29A at ~836bp.
    • Topocloned Samantha Lee's PCR product #1 of rd29A
    • Streaked single colonies of rd29A (Topo Vector)
    • Cell cultured single colonies of rd29A (Topo Vector)
    • Minipreped rd29A (Topo Vector) and digested with EcoR 1 and Pst 1.
  • Matthew
    • Completed ligation and began transformation for 35S-GFP candidates
    • Screened 35S-GFP candidates on chloramphenicol and kanamycin
    • Cultured,miniprepped,digested, and ran on gel 20 candidates
      • One candidate possible for sequencing
Polasko 35sgfp.jpg

 

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

From Left: Lane 1 - 1 kb ladder.

Lane 2 - Uncut 35S-GFP candidate 1

Lane 3 - 35S-GFP candidate 1

Lane 4 - Uncut 35S-GFP candidate 2

Lane 5 - 35S-GFP candidate 2

Lane 6 - Uncut 35S-GFP candidate 3

Lane 7 - 35S-GFP candidate 3

Lane 8 - Uncut 35S-GFP candidate 4

Lane 9 - 35S-GFP candidate 4

Lane 10 - 35S-GFP candidate 5

 

 

    • Submitted for sequencing 1 35S-GFP candidate

Week of October 10-16:


  • Elaine
    • Ran rd29A (Topo Vector) gel.

Rd29A in TOPO Vector.jpg

  • 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).
    • Digested rd29A (Topo Vector) and RFP (pSB1C3)with EcoR1 and Pst 1.
    • Ethanol Precipitation of the rd29A (Topo Vector) and RFP (pSB1C3).
    • Ligation of the rd29A (Topo Vector) and RFP (pSB1C3).
    • Transformation of the rd29A (Topo Vector) and RFP (pSB1C3) to transform to what we want: rd29A (psB1C3 Vector).


  • Christian
    • Transformed ligation product into Top 10 cells using Chloramphenicol and Kan selection
    • 5 colonies selected and inoculated overnight at 37C
    • Miniprep 5 cultures and digested with EcoR1 and Pst1
    • Ran digests on 0.8% agarose gel

DREB1C gel 3.jpg

  • Expected bands appeared at ~500 bp for sample 3 and sample 5
    • Samples DREB1C_2.3 and DREB1C_2.5 selected and sent for sequencing
    • Sequencing data for DREB1C_2.3 had 0 mismatches and was selected
  • Matthew
    • Sequencing results came back positive for 35S-GFP
    • Miniprepped 35S-GFP and made glycerol stock
  • Richard and Nick
    • 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



Week of October 17-23:

  • Elaine
    • 30 Cell cultures of rd29A (pSB1C3)
    • Minipreped 30 cell cultures expected to have the rd29A (pSB1C3). Concentrations were too low and therefore only 4 colonies were digested.
    • Digested 4 colonies (labeled as seen on the gel below: #1, #3, #13, #15) suspected to have the rd29A in the iGEM(pSB1C3) Vector
    • These 4 colonies were ran on a 1.2% Agarose gel for analysis - "rd29A in iGEM (pSB1C3) Vector"

Rd29A in pSB1C3 - Epic Failure.jpg

  • 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.
  • 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.
    • Ran 45 Colony PCR samples expected to have the rd29A in the iGEM(pSB1C3) Vector on 1.2% Agarose gels

Colony PCR -21 - rd29A in pSB1C3 Vector.jpg

  • 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).
    • Minipreped 4 samples of the Colony PCR #21 (labeled 21-1, 21-2, 21-3, 21-4) and streaked them on LB-Chloramphenicol plate.
    • Saved glycerol stocks for 21-3 & 21-4 of the rd29A in pSB1C3 & placed in -80C freezer.
    • Sequenced Colony PCR #21 at the Nevada Genomics Center.
    • Minipreped 1 sample of the 1-9 rd29A + RFP in pSB1C3.
    • Saved 2 glycerol stocks for the 1-9 rd29A + RFP in pSB1C3.
  • Christian
    • Sequencing of DREB1C_3 in pSB1C3 had 0 mismatches and was submitted as part BBaK414007
    • 24 colonies selected from DREB1C/YFP transformation and run in colony PCR using custom DREB1C primers
    • Products of PCR reaction run on a 0.8% agarose gel

DREB1C gel 4.jpg

  • Band at ~500 bp for colony 24
    • Colony 24 was the only positive result
    • Colony 24 DREB1C/YFP construct was sent for sequencing
  • Matthew
    • Submitted 35S-GFP to iGEM
  • Richard and Nick
    • Performed diagnostic digests on fragments that could potentially be Cd2+ promoter; no fragments yielded positive digestion patter...back to the drawing board


Week of October 24-30:


  • Elaine
    • Added rd29A (pSB1C3) into the registry
    • Submitted rd29A (pSB1C3) through the mail.


 

 


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