Colony PCR

Colony PCR is useful for quickly screening your cloning efforts without having to do a mini-prep or lysogen preparation. Bacterial cells are lysed within the PCR tube.

Contents

1. PCR Mix
2. Protocol
3. Notes

1. PCR Mix

Make up the following mix in one tube, then aliquot into individual PCR tubes. The volumes given are for one 25 ul PCR reaction, so you need to multiply by the number of tubes, plus 1 (or 2) to account for pipetting errors.

For example, if you want to screen 10 colonies, make 250 uL total PCR reaction mix

• 5 uL 5x Crimson Taq Buffer (NEB)
• 0.5 uL 10 mM dNTPs
• 0.5 ul forward primer (10 uM)
• 0.5 ul reverse primer (10 uM)
• 18.5 ul water
• 0.125 ul NEB Crimson Taq polymerase

Do not make up more than you need for the current PCR analysis.

2. Protocol

1. Start 1 ml minipreps for each colony that you want to test. Do this as early as possible in the morning. Let them grow until you can see the bacteria, but you don’t need a dense solution. Two-three hours seems to work well.

2. Pipet 20-25 ul PCR master mix into PCR tubes. Add 1 ul growing culture. Save the cultures!

3. Run this PCR program:

   1. 96°, 1 min; 50°, 30 sec; 68°, X min; 10X
   2. 94°, 30 sec; 50°, 30 sec; 68°, X min; 20X

   The extension times should be adjusted based on the length of your expected product (~1 min/kb for Crimson Taq PDF). Note that the the first 10 cycles take the place of a “boil” program, at the expense of polymerase inactivation. Adjust the annealing temperature (50oC) in program as necessary for your primers.

4. Add 5 ul 5X or 6X loading buffer to each tube and run an agarose gel, loading ~10 ul per lane. If using CrimsonTaq buffer, just load 10 ul of PCR reaction directly onto agarose gel.

5. Use the cultures corresponding to PCR-positive colonies to start mini/midi-preps (or whatever prep is appropriate).

3. Notes

I recommend CrimsonTaq DNA polymerase (NEB) for PCR reactions that will be loaded directly onto a gel for analysis.