Endy:Colony PCR: Difference between revisions
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**72 C for X s (elongation) | **72 C for X s (elongation) | ||
*72 C for 10 minutes (final elongation) | *72 C for 10 minutes (final elongation) | ||
*4 C forever | *4 C forever | ||
*For long amplicons, X = 1 minute + 2.5 s per 100bp | *For long amplicons, X = 1 minute + 2.5 s per 100bp | ||
*For shorter amplicons, under ~1kb, this can be shortened judiciously. | *For shorter amplicons, under ~1kb, this can be shortened judiciously. |
Revision as of 09:22, 14 July 2005
See Colony PCR for general information about this protocol and other variants
Protocol
- Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.
Reaction Mix
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
PCR protocol
- 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
- For long amplicons, X = 1 minute + 2.5 s per 100bp
- For shorter amplicons, under ~1kb, this can be shortened judiciously.