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

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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: 55oC (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 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 (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.