Colony PCR

Materials and Reagents

Mix (for 1 tube/1 colony)

• _μL of distilled water (to 50 uL total Volume)

• 5 μL of Taq 10 x buffer

• Optional 5 μL of Taq Mg Stock

• 1 μL of 10mM dNTPs

• 1 μL of 25 μM forward primer

• 1 μL of 25 μM reverse primer

Reference

• NEB Taq DNA Polymerase with Standard Taq Buffer

Procedure

1. Pick 3-10 colonies in total to test, depending on the number of background colonies on your no ligation control plate.

2. Mark each colony (1,2,3…), so you know which one to use if it is successful.

3. Take 3-10 PCR tubes, mark each tube (1,2,3…).

4. Prepare the solution.

5. Pick a single colony with a sterile pipette tip and swirl in a small amount of the mixture (~49 uL). One colony per tube.

6. Boil at 95 degrees Celsius for 20 minutes (Use PCR machine).

7. Spike in 0.5-1.0 μL of Taq Polymerase in each tube, and carry out normal PCR conditions thereafter.

Notes and Cautions

• The more background, the more colonies you will need to screen.

• The website link for description of the Taq Buffer is above. Check for the presence of MgCl2, add if needed.

• Cheap polymerases are preferred, particularly homemade polymerases, to decrease the cost of the screening.