Revision as of 08:01, 18 September 2008

Retransformations 9/15

P126 and 127 retransformed into DH5α. Split 30μL and rest onto 2 diff CM plates.

9/16: Retransform went fine, although still fewer cells/DNA could have been used. Colonies picked for overnight cultures.

P63 EX (8/14, dephos) with:

P97 SP (dephos) with:

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.

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.

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

0 colonies (on KAN)

The following digests were performed using the Fermentas protocol for 25 min: mtrB TOPO ES; mtrB TOPO XP; P63 EX; P97 SP, P101, 102, 116, 117 ES

Aside from 116, 117, the bands were extracted using a clonewell gel. 116 and 117 were not visible.

P63, P97, and P101 were dephosphorylated according to the NEB instructions using Antarctic phosphatase, and the phosphatase was heat killed.

The following ligations were performed (using 1:6 vector:insert ratio):

The DNA was mixed, heated to 65°C for 2 minutes, and then mixed with ligase.

With Takara kit, 6μL DNA mix was added to 6μL ligation mix
With NEB kit, 9μL DNA mix was added to 1.1μL T4 ligation buffer and 1μL T4 ligase

The mix was cooled to 16°C for 45min and then held at 4°C prior to transformation.

@@ Line 37: / Line 37: @@
 Aside from 116, 117, the bands were extracted using a clonewell gel. 116 and 117 were not visible.
-P63, P97, and P101 were dephosphorylated according to the NEB instructions, and the phosphatase was heat killed.
+P63, P97, and P101 were dephosphorylated according to the NEB instructions using Antarctic phosphatase, and the phosphatase was heat killed.
+==Ligations 9/18==
+The following ligations were performed (using 1:6 vector:insert ratio):
+*mtrB TOPO ES + P63 EX
+*mtrB TOPO XP + P97 SP
+*P101 ES + P102 ES
+The DNA was mixed, heated to 65°C for 2 minutes, and then mixed with ligase.
+* With Takara kit, 6μL DNA mix was added to 6μL ligation mix
+* With NEB kit, 9μL DNA mix was added to 1.1μL T4 ligation buffer and 1μL T4 ligase
+The mix was cooled to 16°C for 45min and then held at 4°C prior to transformation.