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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 Gel analysis of GFP in PSB1C3 vectors digested with EcoR1 & PstI

Week of August 22-28: