Matt Gethers/CRI, Thailand/Labwork/PCRs/Screening for Presence of Lox/Gen Cassette in pKn004

=Screening for Insertion of Lox/Gen Cassette in pKn004 (pKn006)=

Rxn Conditions
Annealing Temperature: 55oC (2.3 degrees below annealing temp of BT1188)

Extension Time: 2 minute 40 seconds (~2.6 kb at 1 kb/min ~2.5 min)


 * Note: If the cassette hasn't been ligated into pKn004, this PCR will yield a product of length ~1500 bp.

Cycle (Taq)

7.31.08
Ran a colony PCR on the sole pKn006.L1 transformant. 10 &mu;l reaction: Used 1 &mu;l colony suspension, 1 &mu;l Taq (too much, but small volume hard to pipette), 0.8 &mu;l M13_for (note I had to make a fresh batch this time, 1 &mu;l supposedly 100 &mu;M stock in 99 &mu;l water), 0.8 &mu;l M13_rev, 0.5 &mu;l dNTPs (again, too much but small volume), 0.5 &mu;l DMSO, 1 &mu;l buffer, 0.5 &mu;l Mg2+, 6 &mu;l water. Used reaction conditions as written in protocol. Gel results here.

8.5.08
Ran colony PCRs on 7 putative pKn006.L2 transformants. Made a master mix and aliquotted to each of 9 tubes, then added 1 &mu;l colony suspension (colony in 25 &mu;l water) to each reaction. Ran PCR according to protocol. Gel results here.