Colony PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
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 11:53, 28 June 2005
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.