Q: Southern Hyb times

Howard T. Petrie h-petrie at ski.mskcc.org
Mon Oct 9 12:33:15 EST 1995


In article <scalzod.1.0008CD76 at fred.fhcrc.org>, scalzod at fred.fhcrc.org wrote:

...I am wondering about the hybridization time for probing southern blots.    
What's the shortest time you've probed, under what conditions and how did
it turn out?

There are nice discussions of all of the details of these issues in both
"Gene Cloning: a laboratory manual" and in Current Protocols in Molecular
Biology.  Basically the biggest problem you have to face is complementary
strand reannealing of probe (assuming that you're using double-stranded
probes).  Since high affinity (i.e., probe - target) interactions should
occur much more rapidly than low affinity (i.e., non-specific) ones,
you're almost always going to be better off with shorter rather than
longer hybridization times.  I routinely use 6-8 hours of hybridization
using 20-25 ng of probe and 1.5-3 micrograms of genomic DNA; at least in
my applications, going longer doesn't give better signal, only higher
background.



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