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

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Contents

Screening for Presence and Directionality of HmgR Upstream Fragment in pUC18

Rxn Mix

Reagent Volume/Amount
DNA Template1.0 μl from Colony Suspension
Primer M13 Rev1.2 μl 1 μM Stock
Primer BT10331.2 μl 1 μM Stock
2x PCR Master7.5 μl
H204.1 μl
Total15 μl
Reagent Volume/Amount
Paeru Genomic DNA Template0.5 μl from So Pa Pan stock
Taq0.5 μl
Primer M13 Rev4 μl 1 μM Stock
Primer BT10334 μl 1 μM Stock
dNTPs1 μl 10 mM Stock
DMSO 2.5 μl
Buffer 5 μl
Mg2+ 2.5 μl
dH2030 μl
Total50 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 Denaturation95oC2 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation94oC30 Seconds
Cyclic Annealing55oC30 SecondsUnsure of best temp
Cyclic Extension72oC50 minute
Repeat Cyclic Steps 35x
Final Extension72oC10 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.

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