southern blot strigency problem
rdej at gsbs3.gs.uth.tmc.edu
Tue Nov 11 16:58:21 EST 1997
I am having a difficult time with my southerns. The
problem is that they are too stringent. I am searching for
a whole gene family which I have fairly good evidence there
are at least five members (GH family in squirrel monkey).
The problem is I am using part of the GH (exon 5) as a
probe, (size 200 bp), and I only get one bad on the
southern (genomic digested with RE). I use the Zeta nylon
membrane and follow manufacturer's instructions. For
probing I use .25 NaHPO4, 7% SDS. I then do a low
stringency wash, 0.1% SDS, 0.1X SSC, for 30'. Each of
these is at 60C. I have tried lower temperatures, 55C, no
difference. I use a p32 labeled probe via PCR
incorporation. I am not sure what other info you need, if
Any ideas? I am trying to get low stringency (more bands!)
but no dice. The band I do get (1) is also fairly weak.
Another problem, the probe is binding to my lambda hind III
ladder. (size 2.3, 2, 4 kb bands). Any idea why this is
so? I haven't checked sequence but i find it very odd that
the probe won't bind to genes with 80% homology or so, but
will bind to the ladder.
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