From OpenWetWareAdd to My Links
See Colony PCR for general information about this protocol and other variants
- Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.
- store the colony resuspension at 4C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate.
Use the following reaction mix for each PCR reaction:
- 1 μl 10x Thermo polymerase buffer
- 1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
- 0.15 μl 40 μM FWD primer
- 0.15 μl 40 μM REV primer
- 0.1 μl Polymerase (taq or vent)
- 6.6 μl H2O
- 1.0 μl template suspension
- 95 C for 6 minutes (disrupt cells, separate DNA)
- Cycle 35 times:
- 95 C for 30 s (melting)
- 53 C (or whatever temperature is appropriate) for 30 s (annealing)
- 72 C for X s (elongation)
- 72 C for 10 minutes (final elongation)
- 4 C forever
- For long amplicons, X = 1 minute + 2.5 s per 100bp
- For shorter amplicons, under ~1kb, this can be shortened judiciously.