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:
Week of May 9-15:
- Bryson
- Ran 0.8% agarose gel of pBIB post-phenol:chloroform cleanup.
- Did minipreps on additional pBIB liquid cultures.
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.
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
Week of May 30-June 5:
- Bryson
- HinD3 digest of uncut sample 3
- Ran 08% 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
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
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
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
Week of June 27-July 3:
Week of July 4-10:
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: