Cell Pop PCR

1. Add 37 uL dH20 to a PCR tube.

2. Wearing gloves take a sterile pipette tip and and lightly touch the colony of interest, a little bit of cells go a long way here so don’t take too much of the colony.

3. Let the tip sit in the dH2O for about 5 minutes.

4. Each reaction will also include:

-5 uL of 10x Sigma Taq buffer

-5 uL of 4 mM dNTP mix

-1.25 uL of each PCR primer, 18 uL stock.

-0.5 uL of Sigma Taq DNA polymerase

5. Make a master mix for setting up >5 samples.  Remove the pipette tips, add the master mix and then add the Sigma Taq.  Spin the tubes down briefly if you’d like.

6. Cycle the reactions as follows:

1.      10 minutes at 95 C

2.      30 sec at 95 C

3.      30 sec at 50 C

4.      1 minute at 72 C for each ~500 bp of expected product, a little more extension can’t hurt, I usually tack on an extra minute above this estimate.

5.      Cycle steps 2-4 about 25-30 times.

6.      10 minutes at 72 C

7.      hold at 4 C