IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week11/Chemical and Light: Difference between revisions
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Meng Xiao He (talk | contribs) (→mtrB) |
Meng Xiao He (talk | contribs) (→mtrB) |
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Mix (split into 11): 247.5μL Platinum supermix, 5.5μL BBpfx, 5.5μL BBsfx, 16.5μL H<sub>2</sub>O | Mix (split into 11): 247.5μL Platinum supermix, 5.5μL BBpfx, 5.5μL BBsfx, 16.5μL H<sub>2</sub>O | ||
Rx: 5min denature, 35 cycles of (45s denaturation at 94°C, 30s annealing at 55°C, | Rx: 5min denature, 35 cycles of (45s denaturation at 94°C, 30s annealing at 55°C, 2m40s at 72°C), 7min final extension, hold at 4°C | ||
==Lac QPI== | ==Lac QPI== |
Revision as of 06:56, 16 September 2008
Retransformations 9/15
P126 and 127 retransformed into DH5α. Split 30μL and rest onto 2 diff CM plates.
Ligations 9/15
- Tried the Takara ligation kit: 1μL vector, 4μL insert, 5μL ligation mix
- Transformed into DH5α
mtrB
P63 EX (8/14, dephos) with:
- mtrB TOPO ES (8/15): 0 colonies (on KAN)
- mtrB BB ES (8/14): 3 colonies (on KAN)
P97 SP (dephos) with:
- mtrB BB XP (8/14): TMTC (on AMP)
- mtrB TOPO XP (8/15): TMTC (on AMP)
It seems like there's something wrong with the P97, so if the colony PCRs indicate there's nothing in the plasmids, the tube'll be tossed.
Colony PCR
The 3 mtrB+63 colonies and 4 of each mtrB+97 colonies were picked for colony PCR:
Mix (split into 11): 247.5μL Platinum supermix, 5.5μL BBpfx, 5.5μL BBsfx, 16.5μL H2O
Rx: 5min denature, 35 cycles of (45s denaturation at 94°C, 30s annealing at 55°C, 2m40s at 72°C), 7min final extension, hold at 4°C
Lac QPI
P3+51 EX (8/3, dephos) with P38 oligo mix (Amy's prep)
0 colonies (on KAN)