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*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50
*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 sterile water.
Revision as of 11:05, 14 July 2005
Colony PCR is can be used after a transformation to screen colonies for the desired plasmid. Primers are used which generate a PCR product of known size. Thus, any colonies which give rise to an amplification product of the expected size are likely to contain the correct DNA sequence.
- Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.
Use the following reaction mix for each PCR reaction:
- 1 uL 10x Thermo polymerase buffer
- 1 uL 10x dNTPs (10x = 2.5 mM each dNTP)
- 0.15 uL 40 uM FWD primer
- 0.15 uL 40 uM REV primer
- 0.1 uL Polymerase
- 6.6 uL water
- 1.0 uL template suspension
- 95 C for 6 minutes (disrupt cells, separate DNA)
- Cycle 35 times:
- 95 C for 30 s (melting)
- 53 C for 30 s (annealing)
- 72 C for X s (elongation)
- 72 C for 10 minutes (final elongation)
- 4 C forever (hold)
- For long amplicons, X = 1 minute + 2.5 s per 100bp
- For shorter amplicons, under ~1kb, this can be shortened judiciously.
Also see Knight:Colony PCR.
- I have compared Taq and Vent using this reaction mix and found that Taq worked better. For very long amplicons, I would recommend using Phusion, which has its own reaction mix. I have not found a difference between Phusion and Taq on amplicons shorter than ~3kb.