Yeast Colony PCR Protocol

Yeast Colony PCR

Yeast Colony PCR Lysis Buffer (1):

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

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.
Pick cells from a yeast colony with a plastic pipet tip into microcentrifuge tubes each containing 15 Î¼l of Yeast Colony PCR Lysis Buffer (do not pick too much, if buffer is cloudy you picked enough cells).
Incubate the suspension at 37°C for 30 min to 60 min.
Add 85 µl of ddwater to bring to 100 microliters.
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.
Place the tubes into a heat block for 10 min at 95-100°C (or use a PCR machine).
Briefly mix the tubes by vortexing.
Pellet the cell debris 1 min at 3,000 G.
Use 2.5 µl lysate for 25 µl volume of PCR reaction.

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.