Yeast Colony PCR
Bruce Goode (2-12-01)
1) Grow fresh colonies of yeast on YPD plate (4 days or fewer since re-streaked).
Synthetic plates can be used, but may not work as well.
2) Using yellow tips, transfer a VERY small amount (barely enough to see) of yeast to
each PCR tube. Remove the yeast from the top of the colony, avoiding any agar
contamination in your PCR reaction.
3) Microwave the tubes containing yeast for 70 sec. on HIGH, with the lids off, then
place tubes on ice.
4) Add these ingredients in a fresh tube on ice to make PCR reaction mix:
per 25µl reaction:
ddH2O
17.0µl
10X Taq Mg-free buffer
2.5µl
25mM MgCl2
2.5µl
2mM dNTP mix
1.0µl
10µM stock oligonucleotides
1.0/1.0µl
5) Turn on the PCR machine and initiate the “single colony PCR” program. Be sure to
enable the heating lid. Allow the first leg of the program to reach 95°C, then push the
“pause” button.
6) Add Taq DNA Polymerase (0.25µl per reaction) to your reaction mix on ice and mix
well. Immediately aliquot 24µl of the PCR reaction mix to each tube on ice, mixing to
resuspend the yeast. Use fresh tips for each reaction. Cap the tubes on ice tightly.
7) Quickly transfer the tubes from ice to the pre-heated PCR machine and close the
heated lid.
8) Press the “pause” button to continue the program. Do NOT press the “proceed” button,
as this will cause the machine to skip the first step.
Program:
Step 1: 5 min. at 95°C
Step 2: 30 sec. at 95°C
Step 3: 30 sec. at 55°C
Step 4: 1 min./Kb at 72°C
Step 5: GO TO STEP 2 (34 times)
Step 6: 5 min. at 72°C
Step 7: 24 hr. at 4°C