Colony PCR

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==Procedure==
==Procedure==
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*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 uL sterile water.
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*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.
===Reaction Mix===
===Reaction Mix===

Revision as of 11:05, 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 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

  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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