nonradioactive screening

Tom Chappell dmcbtch at ucl.ac.uk
Sun Jul 17 05:51:11 EST 1994


In article <2vuuqa$6bv at nntp2.Stanford.EDU>
btoomey at leland.Stanford.EDU (Barbara Holland Toomey) writes:

> I getting ready to screen a cDNA library with a DNA probe. I am interested
> in nonradioactive methods to label this probe (I have a 200bp PCR product) 
> since I would like to have kids someday. I am considering Amersham's ECL 
> 3'-oligolabelling system, but I have no experience with any of the 
> nonradioactive methods. I would greatly appreciate any advice on this kit or 
> any others that might be better. Thanks!!!
> Barbara Toomey

I've done 230 bp PCR'ed pombe probes onto P1 and cosmid gridded pombe
libraries with ECL. I get a good positive signal after 2 min exposure
if probe is labelled with random oligo. Signal is much weaker with FITC
(?-I think ECL is anti FITC..) incorporation during PCR. I've used it
as an experiment in the CSH pombe course and it is basically foolproof.
The only problem I run into is getting enough background to actually
see the grid pattern on the filters...

Tom Chappell
MRC Molecular Cell Biology
University College London   



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