Team:Nevada/Notebook

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Team Nevada Notebook

Week of April 11-17:

  • Bryson, Michael, Senny, Tyler
    • Made tobacco cell (NT-1) media in Dr. Shintani's lab

Week of April 18-24:

  • Bryson
    • EcoRI digest of pBIB
    • Made Na acetate buffer

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:


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  • 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:

  • 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:

  • 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:

  • 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

Week of May 30-June 5:

  • 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:

  • 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
    • 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:

  • 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:

  • 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

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
      • PCR results are good for mCherry and EYFP, NOS is suspect
    • Performed transformation protocol with TOPO vector
    • Performed screening on Kan and miniprepped

Week of July 25-31:

  • Elaine
    • GFP Transformation
    • Cell Culture of GFP
    • Miniprep of GFP
    • EcoR I & Pst Digest of GFP
    • Ran 1.2% agarose gel analysis of the GFP digest
    • 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


Week of August 1-7:

  • Elaine
    • Ran 1.2% Agarose Gel analysis of GFP PCR product
    • 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
    • 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
  • 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:

  • 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

Week of August 29-September 4:

  • Elaine
    • Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)
    • 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
    • Analysis of sequence of GFP (PSB1C3)
  • 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

Week of September 12-18:

  • 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

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
    • 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

Week of September 26-October 2:

  • 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
    • 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:

  • Elaine
    • Topocloned PCR product of RD29A
    • Streaked single colonies of RD29A (Topo Vector)
    • Cell cultured single colonies of RD29A (Topo Vector)
  • 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
    • Submitted for sequencing 1 35S-GFP candidate

Week of October 10-16:

  • Matthew
    • Sequencing results came back positive for 35S-GFP
    • Miniprepped 35S-GFP and made glycerol stock

Week of October 17-23:

  • Matthew
    • Submitted 35S-GFP to iGEM


Week of October 24-30:
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