scientificprotocols authored about 8 years ago
Author: Matt Lewis
This is the fastest way to screen bacterial colonies. Our PCR machine takes 24 tubes so I routinely screen 22 colonies + 1 negative + 1 positive control.
Ideally you want a primer pair that can only work if the correct construct is present eg. a vector flanking primer and a gene specific primer. However, this may not allow you a positive control (essential) so you might have to use both vector flanking primers instead. If you have enough space in your PCR machine you could do both.
Add 15 µl of pre-mix to each PCR tube.
This PCR should take less than 2 hours. While the PCR is running prepare the agarose gel ready to analyze the PCR products.
When you have the result you can go to the replica agar plate on the same day and set up miniprep cultures of the likely candidate colonies.