Colony PCR
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===Reaction Mix=== | ===Reaction Mix=== | ||
Use the following reaction mix for each PCR reaction: | Use the following reaction mix for each PCR reaction: | ||
| - | *1 | + | *1 μl 10x Thermo polymerase buffer |
| - | *1 | + | *1 μl 10x dNTPs (10x = 2.5 mM each dNTP) |
| - | *0.15 | + | *0.15 μl 40 uM FWD primer |
| - | *0.15 | + | *0.15 μl 40 uM REV primer |
| - | *0.1 | + | *0.1 μl Polymerase |
| - | *6.6 | + | *6.6 μl water |
| - | *1.0 | + | *1.0 μl template suspension |
===PCR protocol=== | ===PCR protocol=== | ||
Revision as of 12:06, 14 July 2005
Contents |
Description
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.
Procedure
- 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 uM FWD primer
- 0.15 μl 40 uM REV primer
- 0.1 μl Polymerase
- 6.6 μl water
- 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 (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.
Notes
- 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.
