Department of Biological Sciences,

1000 Hilltop Circle

Baltimore, MD 21250

Colony PCR

1. Prepare a master mix containing the following:

1X reaction:

• 2.5 µl 2 mM dNTPs
• 2.5 µl 10 µM primer 1
• 10 µl 2.5X cresol red loading dye
• 2.5 µl 10X PCR buffer
• 5.0 µl sterile water
• 0.5-1 µl Taq polymerase

Note: If using PuReTaq Ready-To-Go PCR Beads (Amersham Biosciences), use 10 µl of water instead of dNTP’s, buffer and enzyme]

To prepare the master mix, multiply the volumes above by the number of colonies to be screened + 1.

2. Aliquot to 0.2 ml labeled PCR tubes and keep on ice.
3. Prepare one selection plate, e.g., LB + ampicillin, for every 20 colonies screened.
4. Label the plate with a grid so that each colony can be associated with a number that matches the number on the PCR tube and can be retrieved once PCR results are known.
5. With a toothpick or sterile loop, pick colonies from transformation plate, patch onto the selection plate and then place remainder in PCR with the same identifier.
6. PCR cycle using the same conditions for the original PCR with those same primers with one modification: include a 5 min 94°C denaturation at the beginning of the cycling reaction.
7. Analyze PCR results by running reactions directly onto an agarose gel (no additional loading dye is required)

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 3/2/2010