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 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.
