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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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


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 cell cultures and nanodrop
    • Ran 1.2% Agarose Gel to check for DNA (GFP in PSB1C3 vector)


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