Probing lambda libraries with oligonucleotides

Steven Goldberg goldberg at bms.com
Thu May 11 08:30:46 EST 1995


I have started to probe a pig liver cDNA library using a labeled
oligonucleotide.  There appears to be inumerable variations on
the different steps of the protocol.  I would be interested to receive
comments or tips on the following steps (especially those that were
successful!):

1.  Transferring and fixing phage DNA:  Some procedures say immerse
the filters in the denaturation and neutralization; others claim this
gives diffuse signals.

2.  End-labeling oligonucleotides:  How much DNA is needed, how much
label (again, I've seen anywhere from 5 t0 25 ul of gamma-dATP rec-
commended).  How about the ECL 3' labeling kit or similar?  

3.  Pre-hybridization and hybridization conditions.  Some use SSPE,
others SSC; some include pyrophosphate; others leave out Denhardt's
in the hybridization.

4.  Wash conditions.  

Thanks for any suggestions.

Steve Goldberg



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