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

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

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)

Run Notes
7.9.08

I ran the protocol using 12 10 &mu;l reactions (6 for pKn003, 6 for pKn004); 9 &mu;l PCR mix and 1 &mu;l 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 &mu;l reactions (7 for pKn003, 8 for pKn004); 9 &mu;l PCR mix and 1 &mu;l 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 &mu;l reactions, all for pKn004. I used 9 &mu;l master mix and 1 &mu;l colony suspension (1 colony in 50 &mu;l water). Used all reaction conditions as written. Gel results here.

Colony PCR Round 2

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