Colony PCR

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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.
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.
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==Procedure==
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==Specific Protocols==
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*[[Endy:Colony PCR]]
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*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.
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*[[Knight:Colony PCR]].
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===Reaction Mix===
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Use the following reaction mix for each PCR reaction:
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*1 μl 10x Thermo polymerase buffer
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*1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
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*0.15 μl 40 μM FWD primer
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*0.15 μl 40 μM REV primer
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*0.1 μl Polymerase
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*6.6 &mu;l H<sub>2</sub>O
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*1.0 &mu;l template suspension
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===PCR protocol===
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*95 C for 6 minutes (disrupt cells, separate DNA)
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*Cycle 35 times:
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**95 C for 30 s (melting)
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**53 C for 30 s (annealing)
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**72 C for X s (elongation)
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*72 C for 10 minutes (final elongation)
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*4 C forever (hold)
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*For long amplicons, X = 1 minute + 2.5 s per 100bp
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*For shorter amplicons, under ~1kb, this can be shortened judiciously.
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Also see [[Knight:Colony PCR]].
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==Notes==
==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.
#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 11:09, 14 July 2005

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.

Specific Protocols

Notes

  1. 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.
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