southern blotting

Joseph C. Bagshaw jbagshaw at wpi.edu
Sat Jul 4 07:30:05 EST 1998


Rob,

You didn't mention what your probe is, but if it contains a highly
repeated sequence, like a microsatellite, it is entirely possible to see
what appear to be smears on a genomic Southern.  Some sequences are so
highly repeated and so widely distributed that they appear to be
ubiquitous.  The distribution of signals is not significantly different
from the distribution of DNA fragments.  I've seen this happen many times.
If this is your problem, you will have to get a different probe.

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.

On 1 Jul 1998, Moens' lab wrote:

> I've tried to do mammalian genomic southerns before but all I get 
> is a smear signal whose intensity correlates with the ethidium bromide 
> intensity in an agarose gel.  Therefore, it is not due to repeat 
> sequences or anything of the sort.  I had PAC DNA on the same southerns 
> and these worked fine.  Also, the lambda marker was clean.  I followed 
> the protocol in Maniatis et al, 1989 "Molecular Cloning" to the letter 
> (for what I can tell) but I keep getting these smears.  I've digested 
> with BamHI to apparent completion, also.  What am I doing wrong?  The 
> amount loaded was about 20 ug and I left hybridizing O/N.  I used 
> Denhardt's solution without formamide.  Does anyone have any idea how I 
> can solve this?
> 
> 	Thank you,
> 	Rob Botelho
> 
> 	yu106715 at yorku.ca
> 	or
> 	moens at yorku.ca
> 
> 




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