Endy:Colony PCR

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.