Background plaques when screening Lambda ZAP II genomic libraries with DNA probe

Michael icarus at uvic.ca
Tue Jan 27 22:48:35 EST 1998


I'm currently have some trouble screening a ZAP II library composed of
genomic DNA of my bacteria of interest. My probe is a 350 bp PCR
product from an insert that I identified using a different vector
system. I didn't gel purify my PCR product, I simply removed the
proteins using a Millilpore spin prep tube and precipitated the DNA.
There should only be 15ng of template amongst 2000ng of PCR product. I
labelled with 32P-ATP using nick translation.


I hybridize O/N at 65C, and wash at 65C twice with 2X SSC, 0.1% SDS,
then four times with 0.2X SSC, 0.1% SDS.  I screened around 36,000
plaques (genome of origin is around 4Mbp). As a +ve control, I dot
44ng of unlabelled probe DNA on Hybond-N+ (all filters used are
Hybond-N+).

The positive control reacts very strongly. I observe no strongly
reacting plaques, but approx. 1/10 of all plaques weakly-mildly react,
The others produce no background at all.

I am wondering if these weakly reacting plaques are possibly due to
labelled template binding and as a result I can't identify true +ves?
Or can excess unincorporated label cause such a problem?

If anyone has any suggestions or has experienced a similar problem in
the past please e-mail me at icarus at uvic.ca

Thanks
Mike



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