southern blot strigency problem

Robert Dejournett rdej at gsbs3.gs.uth.tmc.edu
Tue Nov 11 16:58:21 EST 1997


All:

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.

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.  

Thanks.

-Rob




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