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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:
- 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.
Following is the Endy:Colony PCR protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi