Bacterial colony screening by PCR¶
Contributed by Matt Lewis <firstname.lastname@example.org>
One of the fastest way to screen bacterial colonies.
With a PCR machine that takes 24 tubes you can routinely screen 22 colonies + 1 negative + 1 positive control.
- Plan your primers.
*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. *
- Set up 24 PCR tubes each containing 5ml H2O.
- Touch a fresh toothpick (or yellow tip) onto a colony, dip it into a PCR tube, then streak it onto a fresh replicate agar plate using a numbered template (that is, all 24 colonies on a single agar plate).
- Repeat this for all colonies.
For negative control use a colony that is negative (or use nothing).
For positive control use a colony that will yield a product with your primers. If you don’t have a +ve colony then use a tiny amount of plasmid DNA
- Incubate the replicate agar plate at 37C overnight.
- Set up an appropriate number of PCR pre-mix as follows (e.g. 25x):
1 x pre-mix||25 x pre-mix -|-|- 11.5ul|H2O|287.5ul 2ul|10 x PCR buffer|50ul 0.6ul|MgCl2 (50mM)|15ul 0.5ul|10mM dNTPs (i.e. a mixture of all four at 10mM each)|12.5ul 0.1ul|primer 1 (100mM)|2.5ul 0.1ul|primer 2 (100mM)|2.5ul 5ul|Template|- ul 0.2ul|cheap Taq|5ul
- Add 15ml of pre-mix to each PCR tube.
- Set up PCR programme as follows:
Cycles|Temperature|Duration -|-|- 1|94C|60 seconds 25|94C|30 seconds 55C|30 seconds 72C|1 minute
- 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.
This method is based, with permission, on an original protocol available here.