McClean:Colony PCR (Quick Load)

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Fast colony PCR protocol for quickly screening many yeast colonies. This protocol uses OneTaq Quick-Load 2X Master Mix (NEB #M0486).

  • Cheap (~$0.40/50μl rxn)
  • Fast to set up
  • Scales down well to small volumes. I've done colony PCR with 25μl, 10μl, and 5μl volumes and saw no difference in efficiency.
  • Load onto gel directly after thermocycling -- the master mix contains 2 dyes, Xylene Cyanol FF and Tartrazine, which run at ~4kb and ~10bp respectively on a 1% agarose gel.
  • Not hot start -- set it up on ice and preheat the thermocycler to 94°C.


  • OneTaq Quick-Load 2X Master Mix with Standard Buffer (NEB #M0486)
  • H2O
  • Primers
  • Colonies
  • Sterile toothpicks


  1. Start the thermocycler protocol (see below) to allow the block to preheat to 94°C.
  2. In a PCR tube, mix the components on ice. For a large screen, set up a master mix then aliquot into PCR tubes.
    For a 25μl reaction:
    • 12.5 μl OneTaq master mix
    • 0.5 μl Primer 1 (200 nM final concentration)
    • 0.5 μl Primer 2 (200 nM final concentration)
    • 11.5 μl H2O
  3. Using a sterile toothpick, pick a colony and gently dip into the reaction until it becomes noticeably cloudy. Don't overdo it, faint cloudiness is enough.
  4. Flick tubes gently to mix and collect with a quick spin
  5. Place cells in preheated thermocycler and press 'Enter' to continue protocol
  6. After thermocycling, load 5 μl directly onto agarose gel for visualization
Thermocycler protocol
Temp (°C) Time Purpose
94 HOLD preheat
94 5 min cell lysis
94 15 sec 30X denaturation
Ta 15 sec annealing
68 1 min/kb extension
68 5 min final extension
4 10 min stop reaction


Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

*Taylor D. Scott (talk) 15:33, 20 August 2019 (PDT): Use the NEB Tm calculator to calculate the annealing temp.

*Taylor D. Scott (talk) 15:33, 20 August 2019 (PDT): I have not tried this with E. coli but it probably will work.


Relevant papers and books


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