Screening a Gene Library - I want to use colony PCR, but How (Jun/09/2006 )

Hello
I have a gene library of around 1250 clones with a large cosmid insert in E.coli. I have a few sequences of the desired gene, initial N terminal and some sequences from within the gene. Does any body know a rapid and effective method to screen all of the clones. I am thinking of doing Colony PCR but I dont want to do all of 1250 PCRs.
Thanks in Advance.

My initial impression (not having screened such a huge library ever) would be to pool an aliquot of all the clones and do PCR with that and use a pure PCR product as a probe for colony hybridization of your arrayed clones. Hope it helps

If you still have the original library clone mixture (not the picked versions) you can do a colony blot, hybridize a probe to the blot, and then sequence (or amplify) the selected clone. The gene should, of course, be present in the genomic DNA of whatever was used to make the library, so you could just PCR out the fragment from that source.

One way is to make pools of you clone with around 50 clones in each pool. You will have 25 PCR to do then. With any of the positive pools you get make sub-pools from these with 5 clones in each. Finally screen the individual clones from the positive sub-pools. This should keep the number of PCR needed to under 40.

Daniel

Reviews of DNA sequencing services