"

From 2010.igem.org

You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.
Your team picture	Team Example

Home	Team	Official Team Profile	Project	Parts Submitted to the Registry	Modeling	Notebook	Safety

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

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

Week of July 11-17:

Week of July 18-24:

Week of July 25-31:

Week of August 1-7:

Week of August 8-14:

Week of August 15-21:

Week of August 22-28: