Colony PCR
From OpenWetWare
(Difference between revisions)
| Line 1: | Line 1: | ||
| + | ==Description== | ||
| + | |||
| + | Colony PCR is generally used after a transformation to screen colonies via PCR for the desired insert. 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 correct ligation products. | ||
| + | |||
| + | ==Procedure== | ||
| + | |||
*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 uL sterile water. | *Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 uL sterile water. | ||
| - | ==Reaction Mix== | + | ===Reaction Mix=== |
Use the following reaction mix for each PCR reaction: | Use the following reaction mix for each PCR reaction: | ||
*1 uL 10x Thermo polymerase buffer | *1 uL 10x Thermo polymerase buffer | ||
| Line 11: | Line 17: | ||
*1.0 uL template suspension | *1.0 uL template suspension | ||
| - | + | ===PCR protocol=== | |
| - | + | ||
| - | ==PCR protocol== | + | |
*95 C for 6 minutes (disrupt cells, separate DNA) | *95 C for 6 minutes (disrupt cells, separate DNA) | ||
*Cycle 35 times: | *Cycle 35 times: | ||
| Line 23: | Line 27: | ||
*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. | ||
| + | |||
| + | 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. | ||
Revision as of 14:53, 28 June 2005
Contents |
Description
Colony PCR is generally used after a transformation to screen colonies via PCR for the desired insert. 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 correct ligation products.
Procedure
- Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 uL sterile water.
Reaction Mix
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
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.
