Hi Background Plague Lifts

Ken Soderstrom soderstk at ucs.orst.edu
Thu Oct 9 13:17:13 EST 1997


Mike:

Are you making your probe by random priming?  What is the source of the
template?

The one case of all-plaque-labeling that I'm aware of was due to the
template used for random primer probe synthesis.  This template was a PCR
fragment generated with vector primers that contained about 50 bp of vector
sequence.  The entire fragment was ~800 bp but those 50 vector bases, under
probably not too stringent conditions, was enough to label every plaque.

Hope you solve your problem,

-- 
Ken Soderstrom
soderstk at ucs.orst.edu


Michael Salvucci <mesalvu at ix.netcom.com> wrote in article
<61j49b$rho at dfw-ixnews8.ix.netcom.com>...
> Dear Netters,
> 
> We are having a problem with very high backgrounds in our plaque lifts.  
The
> problem is that all of the plaques are hybridizing with our probe.  The
> problem occurs with both DNA and riboprobes for different genes, even
though
> we are using standard and even strigent conditions for prehybridization,
> hybridization and washing.  We are using a library prepared in the
Lambda-ZAP
> Express phagemid and infecting a bacterial lawn of XL-1 Blue cells.  Any
> suggestions would be greatly appreciated.  Please email me directly. 
> 
> Thanks........MIKE
> 



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