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 Mix

Reagent	Volume/Amount
DNA Template	1.0 μl from Colony Suspension
Primer M13 Rev	1.2 μl 1 μM Stock
Primer BT1033	1.2 μl 1 μM Stock
2x PCR Master	7.5 μl
H20	4.1 μl
Total	15 μl

Reagent	Volume/Amount
Paeru Genomic DNA Template	0.5 μl from So Pa Pan stock
Taq	0.5 μl
Primer M13 Rev	4 μl 1 μM Stock
Primer BT1033	4 μl 1 μM Stock
dNTPs	1 μl 10 mM Stock
DMSO	2.5 μl
Buffer	5 μl
Mg2+	2.5 μl
dH20	30 μl
Total	50 μl

Rxn Conditions

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

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

Cycle (Taq)

Step	Temperature	Duration	Notes
Initial Denaturation	95oC	2 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation	94oC	30 Seconds
Cyclic Annealing	55oC	30 Seconds	Unsure of best temp
Cyclic Extension	72oC	50 minute
Repeat Cyclic Steps 35x
Final Extension	72oC	10 minutes

Run Notes

6.30.08

I ran 7 colony PCRs and 1 semi-positive control using the Taq protocol on DH5α transformed with pKn002. I used primers M13_for and BT2696 with a T_m 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 (DH5α 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 μl template (0.5 μl genomic temp for positive control) in 10 μl reactions. Followed the reaction mix and protocol as written otherwise.