Debugging PCRs with Bn PUM1 F2, Mm MAEL F2, Mm PUM, Mm TDRD1

11/01/12

RACE PCR: Bn PUM1 F2, Mm MAEL F2

PCR: Mm PUM, Mm TDRD1

BnMmPCRs 2012-01-11 19hr 04min

I cut the bands from the RACE PCR very quickly under UV light (<10s each) and purified regular PCR directly (band was not cut from the gel). This is to be sure that exposure to UV is not damaging the samples, which could be responsible for the ligation failures.

Standard ligation set up at 21:00 (protocol values).

12/01/12

Heat shock transformation and plating at 17:30, looking good.

13/01/12

Bn PUM1 F2 and Mm MAEL F2 did not have many colonies (6 and 3, respectively). The latter could be because of the amount of PCR product interfering in the ligation. Mm PUM and Mm TDRD1 had extra colonies and I included 6 of each in the colony check PCR, started at 10:50.

MmColony 2012-01-13 15hr 17min

Positive colonies! Looks like purifying PCR product directly is more worth it, if there is only one band. Apparently the UV was indeed damaging the DNA from the samples. Asterisks marks are colonies to be picked for the MiniPreps.

Bn PUM1 F2 No positive colonies, only a 500 bp one.
Mm MAEL F2 Positive: 1, 2, 3
Mm PUM Positive: 3, 4, 6
Mm TUDOR Positive: 3, 4, 5 (2 also, but bit shorter)

15/01/12

Add 3 mL of LB to MiniPrep tubes and leave in the shaker.