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Yeast Colony PCR

    Yeast Colony PCR Lysis Buffer (1):

    0.1 M NaPO4 buffer, pH 7.4
    4800 U/g Lyticase


  1. Add 11 mg Lyticase in 1 ml NaPO4 buffer; alternatively, touch the lyticase powder with a 1 ml pipet tip and resuspend it in 1 ml of buffer. Use freshly prepared.
    Zymolyase (60 U/ml) can replace Lyticase.
  2. Pick cells from a yeast colony with a plastic pipet tip into microcentrifuge tubes each containing 15 µl of Yeast Colony PCR Lysis Buffer (do not pick too much, if buffer is cloudy you picked enough cells).
  3. Incubate the suspension at 37°C for 30 min to 60 min.
  4. Add 85 µl of ddwater to bring to 100 microliters.
  5. Add ~25 µl of glass beads (0.5 mm size) into each tube, mix by vortex for 1 min.
    You can omit this step, however it will increase lysis efficiency. This step becomes more important if your lyticase or zymolase treatment is not optimum.
  6. Place the tubes into a heat block for 10 min at 95-100°C (or use a PCR machine).
  7. Briefly mix the tubes by vortexing.
  8. Pellet the cell debris 1 min at 3,000 G.
  9. Use 2.5 µl lysate for 25 µl volume of PCR reaction.

    10. Run the following PCR program:
    Step 1: 94 °C, 2 min 30 sec
    Step 2: 94 °C, 30 sec
    Step 3: 56 °C, 30 sec
    Step 4: 72 °C, 1 min per kb
    Step 5: repeat from step 2, 33 cycles
    Step 6: 72 °C, 10 min

    PCR reaction:
    ddwater: 14.65 microliters
    10x Buffer (MgCl2-) : 2.50
    50 mM_MgCl2- : 0.75
    2 mM dNTP : 2.50
    10 uM primer : 1.00
    10 uM primer : 1.00
    Platinum Taq Pol : 0.10 (0.5U/25 microliter rxn)
    Genomic DNA : 2.50 (for 25 cycles)
    Total : 25.00 microliters.

As a general rule

(1) Iyer K. et al. Sci. STKE, 15 March 2005, Vol. 2005, Issue 275, p. pl3.

Posted by Erbay Yigit on 14.May.2008. Last modified on 02.May.2010.