Endy: Colony PCR
General Information
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.
ProtocolUse a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water. store the colony resuspension at 4C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate. Reaction MixUse the following reaction mix for each PCR:
· 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 (or whatever temperature is appropriate) 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 horter amplicons, under ~1kb, this can be shortened judiciously.
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.
Fuente: http://openwetware.org/wiki/Endy:Colony_PCR
General Information
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.
ProtocolUse a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water. store the colony resuspension at 4C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate. Reaction MixUse the following reaction mix for each PCR:
· 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 (or whatever temperature is appropriate) 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 horter amplicons, under ~1kb, this can be shortened judiciously.
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.
Fuente: http://openwetware.org/wiki/Endy:Colony_PCR
