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cDNA gt11 screening

P.F. Watson mbpfw at seqnet.daresbury.ac.uk
Fri Aug 27 05:47:51 EST 1993

Hi all

We are currently screening  human ovarian cDNA library in gt11.
We thought that as a method of checking the library quality and immuno-
screening method it might be interesting to screen with an anti-actin
antibody. Of course there are a lot of actin isoforms so I tried a polyconal
anti-actin serum from Sigma (A2668). And so far have got ZIP! from about 60,000
plaques and HRP-conjugated secondary Ab with chloro-napthol.
The library is from Clontech and I have been trying to get from them the fre    
frequency of actin inserts fro this library which they obtain as part of their 
routine quality assurance procedure for each batch (I am told).
I know people generally do DNA hybridisation for determining actin insert freq      
frequency and we are also in the process of sizing random clones as well but
it would have been nice to have had some positives using antibody screening.

Has anybody else tried this? or had similar experiences. Better still can an      
suggest a good antibody against an abundant human cellular constituent that
we could try?


Phil Watson
Department of Medicine
Northern General Hospital
mbpfw at seqnet.dl.ac.uk

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