PCR of cDNA library

john brennand john.brennand at gbapr.zeneca.com
Mon Apr 22 05:15:01 EST 1996


Mitch

We've done this succesfully a couple of times.  

What worked best in our hands was:-  

Plate the library out onto 20 plates @ 25-50,000/plate.  Make 20 lysates. 
Boil, for 2', aliquots of the lysates. Do the primary PCR with a lambda 
primer from each side of the cloning site and your specific primer - ie 2 
PCR's per lysate (only need to do 2 PCR's if the library is 
non-directionally cloned).  Run the gel, blot and probe with an upstream 
specific primer (not the PCR primer!).  If nothing is positive, do a 
second PCR on 1 ul of a 1:100 dilution of each sample, using the same 
lambda primer but with a nested or upstream gene specific primer.  Gel, 
blot & probe.  Identify the lysate giving the longest product, plate out 
and, either repeat the PCR screening, or screen by hybridisation with 
your specific PCR product.

Hope this helps,

John





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