How do you reduce backgrounds...?

John Watson ajwatson at romeo.caltech.edu
Mon May 23 13:04:41 EST 1994


In article <94143.084715PXH at psuvm.psu.edu>, Deepti Pradhan
<PXH at psuvm.psu.edu> wrote:
> 
> I would really appreciate any suggestions from nettors on how I could reduce
> the nonspecific signals I am getting in my cDNA library screening.  I should
> mention that my hybridization and washes are at 68o [SSC protocol, no
> formamide here].  I did not have this problem before, when I was using an
> oligo probe.  The first time I saw this I thought my DNA was not clean , so
> I cut it again [it's in bluescript], extracted, made fresh probe and I
> *still* see everything light up.  Since it is a primary screen, it's got to
> be nonspecific signals.  BTW the library is in gt11.  This is the first time
> I am doing this stuff, so *any* [constructive :)] suggestions would be
> appreciated!
> Thanks,
> Deepti

If you are not gel-purifying your probe DNA away from the vector, you
should do so.  It's my guess that the background is cross-hybridization
between the plasmid and phage sequences.  Even though gel purification
cannot absolutely remove every last trace of vector sequence, it vastly
diminishes it.  Another probe preparation method that works well for me
(even when screening lambda ZAP libraries with probes cloned into
Bluescript vectors) is to PCR amplify some or all of the insert (I usually
do about 300-400 bp) from a *small* amount of recombinant plasmid, then
gel-purify.

John Watson
Caltech Division of Biology, 147-75
Pasadena CA 91125
ajwatson at romeo.caltech.edu



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