Endy:Colony PCR protocol

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Solutions/reagents:

  • Thermo polymerase buffer
  • dNTPS
  • Forward primer
  • Reverse primer
  • Taq or Vent Polymerase
  • Template suspension
  • H2O

Equipment:

  • Thermocycler
  • Reaction tubes

Steps:

  1. Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 ?l sterile water.
  2. Store the colony resuspension at 4°C 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.
  3. Reaction mix
    Set up a reaction in reaction tube (1) as follows on ice:

     Initial concentrationFinal concentrationFinal volume per 10 µl reaction
    Thermo polymerase buffer10X1X1 µl
    dNTPS10X1X1 µl
    Forward primer40 µM0.6 µM0.15 µl
    Reverse primer40 µM0.6 µM0.15 µl
    Taq or Vent Polymerase----0.1 µl
    Template suspension----6.6 µl
    H2O----1 µl
  4. PCR protocol
    Program a standard thermocycler to run the reaction using the following parameters:
    Initial denaturation
    • Denature: 95°C, 6 mins
    Thermocycling
    • No. of cycles: 35
    • Denature: 95°C, 30 secs
    • Anneal: 53°C, 30 secs
    • Elongate: 72°C, 60 secs
    Termination
    • Elongate: 72°C, 10 mins
    • Hold: 4°C, until removed from machine
    For long amplicons, elongation time = 1 minute + 2.5 s per 100bp.
    For shorter amplicons, under ~1kb, this can be shortened judiciously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 28 mins

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