Blackburn:Yeast Colony PCR

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Revision as of 21:21, 11 June 2006 by Tetm2002 (talk | contribs) (Notes)


This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.



Yeast Cell Lysis

  1. Aliquot 3uL of 0.02M NaOH into PCR tubes.
  2. Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
    • If the solution is cloudy, you've added enough cells.
  3. [optional] Quick-freeze the cells in liquid nitrogen
    • I normally skip this step. Works just fine.
  4. Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes.
    • In the mean time, prepare the master mix for the PCR reaction.
    • The boiled samples are stable at room temp for sometime. Keep on ice or freeze for longer.


  1. Prepare the master mix solution containing:
    • 5uL 5X Q-solution
    • 2.5uL 10X PCR Buffer
    • 0.5uL dNTPs (10mM each)
    • 0.5uL foward primer (100uM)
    • 0.5uL reverse primer (100uM)
    • 0.25uL Taq
    • 12.75uL ddH2O
  2. Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume).
  3. Run the following PCR cycle:
    1. 5 min at 94C
    2. 30 cycles of:
      1. 30 sec at 94C
      2. 30 sec at 55C (or appropriate annealing temperature)
      3. 1 min/kbp at 72C
    3. 10 min at 72C


  • The PCR product can be loaded onto agarose gels directly without addition of loading buffer.
  • For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume.


Relevant papers and books

  1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. PubMed ID:6947258 | HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. PubMed ID:13718526 | HubMed [Jacob-JMB-1961]
  3. ISBN:0879697164 [Ptashne-Genetic-Switch]

All Medline abstracts: PubMed | HubMed


  • Who has experience with this protocol?