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 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: 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)
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 | 1 minute 50 seconds | |
Repeat Cyclic Steps 35x | |||
Final Extension | 72oC | 10 minutes |
Run Notes
7.9.08
I ran the protocol using 12 10 μl reactions (6 for pKn003, 6 for pKn004); 9 μl PCR mix and 1 μl 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 μl reactions (7 for pKn003, 8 for pKn004); 9 μl PCR mix and 1 μl 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 μl reactions, all for pKn004. I used 9 μl master mix and 1 μl colony suspension (1 colony in 50 μl water). Used all reaction conditions as written. Gel results here.
Colony PCR Round 2
I ran the protocol using 12 10 μl reactions using only colonies from the pKn004.L3 plate. I used 9 μl master mix and 1 μl colony suspension (1 colony in 50 μl water). Used all reaction conditions as written. Gel results here.