Colony PCR

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(Reaction Mix)
(Reaction Mix)
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*1 μl 10x Thermo polymerase buffer
*1 μl 10x Thermo polymerase buffer
*1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
*1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
-
*0.15 μl 40 uM FWD primer
+
*0.15 μl 40 μM FWD primer
-
*0.15 μl 40 uM REV primer
+
*0.15 μl 40 μM REV primer
*0.1 μl Polymerase
*0.1 μl Polymerase
-
*6.6 μl water
+
*6.6 &mu;l H<sub>2</sub>O
*1.0 &mu;l template suspension
*1.0 &mu;l template suspension

Revision as of 12:07, 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 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
  • 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 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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