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Colony PCR

The 1st person in this protocol is Dr. Stacey Smith, Ph.D.; the 2nd person is you.

This is but one way of doing colony PCR. I have written it with Qiagen reagents but would look similar with other brands (just be sure the buffer matches the TAQ you use).

In each PCR tube goes:

1.25ul 10x PCR buffer
1.25ul Q-Solution (that mysterious PCR additive which Qiagen provides)*
0.50ul MgCl2 (25mM)
0.25ul dNTPs (10mM)
0.25ul forward primer (M13F or SP6 with PGEM vector)
0.25ul reverse primer (M13R or T7 with PGEM vector)
0.06ul Qiagen TAQ polymerase
8.19ul H2O
12.5ul total

After aliquoting the master mix into each tube, add the template by gently touching a tip to the colony of choice and ejecting the tip into the tube. No need to get a lot of the colony and be careful not to scrape up agar. When each tube has a tip in it, give the plate a little wiggle and then pull out the tips, disposing them in the biohazard. Add the caps and run the COLONY program:

95 C 5 min
Then 30 cycles of:
95 C 30s
53 C 30s
72 C 1min30s**
72 C 5min

*My opinion is that the Q-solution keeps all the bacterial funk from inhibiting the PCR so don't skip it!
**You could make the program faster by shortening this if your product was expected to be small. Rule of thumb is 1 minute per 1kb of product.

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