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

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

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: 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)

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 2 minute 40 seconds
Repeat Cyclic Steps 35x
Final Extension 72oC 10 minutes

Run Notes

7.31.08

Ran a colony PCR on the sole pKn006.L1 transformant. 10 μl reaction: Used 1 μl colony suspension, 1 μl Taq (too much, but small volume hard to pipette), 0.8 μl M13_for (note I had to make a fresh batch this time, 1 μl supposedly 100 μM stock in 99 μl water), 0.8 μl M13_rev, 0.5 μl dNTPs (again, too much but small volume), 0.5 μl DMSO, 1 μl buffer, 0.5 μl Mg2+, 6 μl 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 μl colony suspension (colony in 25 μl water) to each reaction. Ran PCR according to protocol. Gel results here.