Endy:Colony PCR

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

Revision as of 08:56, 13 June 2007

See Colony PCR for general information about this protocol and other variants

Protocol

  • Use 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 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 (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 shorter amplicons, under ~1kb, this can be shortened judiciously.
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