Bacterial Colony PCR

Objective:

This protocol allows rapid detection of transformation success when primers are available to allow determination of correct ligation products by size or hybridization.

Procedures:

Start => Bacterial colonies from transformation.
Prepare 2mL culture tubes w/ an appropriate selection media for your plasmid of interest, label 1.5mL tubes and place inside culture tube as a cap.

Prepare PCR master mix: buffer, MgCl2, dNTPs, primers, sucrose red, and enzyme- Aliquot 25µL of master mix into a PCR tube for each colony to be picked. (See PCR .html">general PCR instructions for more instructions on how to set up the PCR reaction.)

Select colonies to pick. Pick colony with a sterile toothpick or pipet tip. Dab the toothpick or pipet tip into the master mix then place the toothpick or pipet tip in a correspondingly labeled culture tube.

Run PCR w/ appropriate conditions for your primers and expected product, Making sure to begin your PCR protocol with an extended time at 95°, (e.g. 5 minutes).

Grow the 2mL cultures O/N at 37°.

Run 10µL of the PCR reaction on an agarose gel to identify which cultures to keep for plasmid DNA isolation.