PCR library using M13 and specific internal primer

john brennand john.brennand at gbapr.pharmaceuticals.zeneca.tmailuk.sprint.com
Wed Jan 3 11:57:25 EST 1996


Marianne

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.

John





More information about the Methods mailing list