Hi Background Plaque Lifts

Geoffrey Kidd GKidd at Aptagen.com
Thu Oct 9 16:35:50 EST 1997


I would bet that it's not a non-specific hybridization, which then means that your plaques contain something that is also in your probe.  
The vector from which you obtain your probe presumably has a drug-resistance gene, as do the XL-1 Blue cells (in the F' episome).  Assuming 
these 2 genes are for the same drug, then using the whole plasmid as the probe would light up all your plaques.  As for the riboprobe, if your 
template is not linearized or incompletely cut, you could get transcriptional readthrough right into the Amp-resistance gene, which would 
again cause all of your plaques to light up.  Try spotting your parent plasmid (lacking your gene of interest) onto a blot and probe.  If this 
is not the answer, it could be that some lambda DNA is in your plasmid.  This happened to me once.  Someone gave me a plasmid to use as a 
probe and didn't tell me that when she had fished the gene out of a lambda library, she excised it using a restriction site *within* lambda, 
so that the gene had a small piece of lambda DNA at the end.  Aside from being a strong argument in favor of legalized strangulation, this 
taught me to distrust and verify (apologies to Mr. Reagan).  Try spotting some Lambda-ZAP Express phagemid DNA on a blot and probing.  Good 
luck!

Geoffrey Kidd


Michael Salvucci wrote:
> 
> 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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