IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week11/Chemical and Light

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

2KB ladder used. Clearly, none of the clones actually had a 2kb+ band, so mtrB was not incorporated.

Lac QPI

P3+51 EX (8/3, dephos) with P38 oligo mix (Amy's prep)

0 colonies (on KAN)