PCR of cDNA library
john.brennand at gbapr.zeneca.com
Mon Apr 22 05:15:01 EST 1996
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,
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