Hi Background Plague Lifts

Dom Spinella dspinella at chugaibio.com
Thu Oct 9 14:58:20 EST 1997


> 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.

Almost certainly, your probe contains some vector-derived sequence that
is hybridizing to the lambda ZAP vector backbone.  Riboprobes run off
from typical T3 or T7 promoters must swim through the polylinker of the
vector before they start copying the insert, and this could easily be a
long enough run of homology to allow hybriridization to all plaques
(lambda ZAP has the same polylinker as pBluescript as I recall).  If you
are using a cloned probe, digest out the insert and separate it from any
vector sequence before labeling.  If you must use a riboprobe, be sure
you generate it in a vector whose polylinker differs from that of lambda
zap. Good luck. -- Dom Spinella



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