Blackburn:Yeast Colony PCR v2.0 protocol

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Solutions/reagents:

Equipment:

  • Thermocycler
  • Sterile 0.6-ml tubes

Steps:

  1. Yeast Cell Lysis

    1. Measure out 10 µl of 0.02M NaOH into sterile 0.6-ml microcentrifuge tube (1).
    2. Add a small colony.
      Resuspend pellet by vortexing/by shaking vigorously.
      If the solution is cloudy, you've added enough cells.
      I have been told adding too much yeast can inhibit the reaction.
    3. Set the thermocycler to run the following program:
      • 99°C, 10 mins
      In the mean time, prepare the master mix for the PCR reaction.
      The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.
  2. PCR

    1. Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (2):

       Q-solutionPCR bufferdNTPsForward primerReverse primerTaqddH2O
      Colony PCR2 µl1 µl0.2 µl0.2 µl0.2 µl0.1 µl5.3 µl
    2. Set aside a fresh sterile 0.6-ml microcentrifuge tube (3). Call it Master Mix aliquot.
      Measure out 9 µl of master mix solution into Master Mix aliquot.
    3. Add 1 µl of boiled sample.
      A multichannel pipette is helpful here.
    4. Program a standard thermocycler to run the reaction using the following parameters:
      Initial denaturation
      • Denature: 95°C, 5 mins
      Thermocycling
      • No. of cycles: 30
      • Denature: 95°C, 10 secs
      • Anneal: 50°C, 10 secs
      • Elongate: 72°C, 60 secs
      Elongation time : 1 min/kbp.I generally do 30 sec elongation.
      Termination
      • Elongate: 72°C, 10 mins
      • Hold: 4°C, until removed from machine
      I don't think this step is critical.

    TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 6 mins

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