Probing lambda libraries with oligonucleotides
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
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.
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