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

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Screening for Presence of HmgR Downstream Fragment in pUC18 (pKn004)

Rxn Mix

Reagent Volume/Amount
Paeru Genomic DNA Template 0.5 μl from So Pa Pan stock
Taq 0.5 μl
M13 for 4 μl 1 μM Stock
BT1188 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 (2.3 degrees below annealing temp of BT1188)

Extension Time: 1 minute 50 seconds (1681 bases at 1 kb/min ~1 min 41 seconds)

  • Note: If the HmgR downstream fragment hasn't been ligated into pKn002, this PCR will yield a product of length ~500 bp.

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
Cyclic Extension 72oC 1 minute 50 seconds
Repeat Cyclic Steps 35x
Final Extension 72oC 10 minutes

Run Notes

7.9.08

I ran the protocol using 12 10 μl reactions (6 for pKn003, 6 for pKn004); 9 μl PCR mix and 1 μl sample. Ran protocol as written; PCR was done jointly with the pKn003 colony PCR. I got product at ~500 bp suggesting the ligation of the fragment into the vector failed and that the vector ligated to itself - that's weird because I digested with SphI and HindIII, which don't leave compatible overhangs. My digest or purification must be inefficient. Gel results are here.

7.15.08

I ran the protocol using 15 10 μl reactions (7 for pKn003, 8 for pKn004); 9 μl PCR mix and 1 μl sample. Ran protocol as written; run jointly with the pKn003 colony PCR. Only bands at ~500 bp showed up again - need to fix digest. Gel results are here.

7.24.08

Colony PCR Round 1

I ran the protocol using 16 10 μl reactions, all for pKn004. I used 9 μl master mix and 1 μl colony suspension (1 colony in 50 μl water). Used all reaction conditions as written. Gel results here.

Colony PCR Round 2

I ran the protocol using 12 10 μl reactions using only colonies from the pKn004.L3 plate. I used 9 μl master mix and 1 μl colony suspension (1 colony in 50 μl water). Used all reaction conditions as written. Gel results here.