low stringency hybridization

bbeitzel at biomail.ucsd.edu bbeitzel at biomail.ucsd.edu
Tue Nov 3 11:57:51 EST 1998


What I usually do is start off with a relatively low temp hybridization,
say 50 degrees C, then wash 2 times at room temp with 2XSSC/0.1%SDS, then
2 times at RT with 0.1XSSC/0.1%SDS.  Place the filters on film and check
the next day.  If there is too much background, increase the temperature
of the 0.1XSSC/0.1%SDS by 5-10 degrees (depending on background) and
repeat the wash.  Put the filters back on film and check again the next
day.  In this way, you can keep increasing the wash temperature until you
reduce your background enough to show possible positive plaques/colonies.

Hope this helps,
Brett Beitzel
bbeitzel at biomail.ucsd.edu

In article <363E07DF.5FD4EC5C at tc.umn.edu>, Guo-Yun Yu <gyy at tc.umn.edu> wrote:

> Does anybody have a protocal for low stringency hybridization screening
> of cDNA library?  I need a protocal that I can easily change the
> striengency.  I have a dna probe which is about 40% identity with the
> target cDNA clone, at protein level.  I think I need a really low low
> stringency hybirdization, and I have to do lots of test using positive
> control.
> Could you suggust to me some protocals?
> Thanks!



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