- Prepare a master mix containing the following:
- 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.
- Aliquot to 0.2 ml labeled PCR tubes and keep on ice.
- Prepare one selection plate, e.g., LB + ampicillin, for every 20 colonies screened.
- 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.
- 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.
- 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.
- Analyze PCR results by running reactions directly onto an agarose gel (no additional loading dye is required)
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