PCR library using M13 and specific internal primer
john.brennand at gbapr.pharmaceuticals.zeneca.tmailuk.sprint.com
Wed Jan 3 11:57:25 EST 1996
Yes, there was a reference in Biotechniques to this approach within the
last year - sorry i've mislaid it.
The gist of what you do - and we have done this succesfully - is to
plate the library out onto 10-20 plates (20-50K/big plate). Scrape off
to make plate lysates. PCR each one with your specific primer and pUC
specific oligo's - will need to use 2 pUC primers (ie 2 PCRs/lysate)
unless your library is directionally cloned. Run out on gel. You usually
see several bands most none specific some hopefully specific. Southern
blot the gel. Probe with a more 5', specific oligo than you used in the
PCR. Identify the PCR giving the biggest, specifically hybridising band.
Replate that lysate at 10 fold less complexity. Repeat the above - or
library screen in the conventional way - until you get a single clone.
More information about the Methods