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 Template0.5 μl from So Pa Pan stock
Taq0.5 μl
M13 for4 μl 1 μM Stock
BT11884 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 (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 Denaturation95oC2 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation94oC30 Seconds
Cyclic Annealing55oC30 Seconds
Cyclic Extension72oC2 minute 40 seconds
Repeat Cyclic Steps 35x
Final Extension72oC10 minutes

Run Notes


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.


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.

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