Matt Gethers/CRI, Thailand/Labwork/PCRs/Screening for Presence and Directionality of HmgR Upstream Fragment in pUC18

=Screening for Presence and Directionality of HmgR Upstream Fragment in pUC18=

Rxn Conditions
Annealing Temperature: 55oC (1.7 degrees below annealing temp of BT2696)

Extension Time: 50 seconds (A little more than 1 minute/kb)

Cycle (Taq)

Run Notes
6.30.08

I ran 7 colony PCRs and 1 semi-positive control using the Taq protocol on DH5&alpha; transformed with pKn002. I used primers M13_for and BT2696 with a Tm of 55 degrees and an extension time of 1:45. The gel didn't turn up any product and upon reviewing the PCR, I realized that the BT2696 binding site is no longer present after the HmgR upstream fragment is cut with PstI, so the PCR couldn't have worked.

7.2.08

I ran the same 7 colony PCRs from the 6.30.08 run (DH5&alpha; transformed with pKn002). For the positive control, I used genomic template and primers BT1032 and BT1033. I only had that brilliant idea after I'd already added M13_rev to the positive control mix, so there's no telling what will happen with that reaction. I used a 55 degree annealing temp, 50 second extension time. I used 1 &mu;l template (0.5 &mu;l genomic temp for positive control) in 10 &mu;l reactions. Followed the reaction mix and protocol as written otherwise.