Help--my genomic DNA is migrating unevenly

Paul Baum baum at mendel.berkeley.edu
Fri Apr 21 04:26:42 EST 1995


I have been doing many Southern blots using C. elegans genomic DNA.  I am
finding that when I use the same enzyme to digest different samples, I get
similar band patterns, but the bands appear to have migrated at different
speeds (i.e. they are not aligned as I would expect, but skewed up or down
in neighboring lanes.)  I thought that this was a problem with excess
salt, but repeated EtOH precipitations, spooling out of DNA and 70% EtOH
washes have failed to solve the problem.

Some other info:
--my comb is not warped
--I don't have any problem getting plasmid digests to migrate evenly
--the genomic DNA prep involves SDS/Proteinase K digestion of worms,
followed by several phenol extractions and then ethanol precipitation. 
All of the samples were prepared in parallel.
--I add spermidine (5mM final conc.) to my digests.  The problem occurs
whether I ethanol precipitate after my digest or not.
--I am using the same concentration and volume of genomic DNA in each digest.
--the gels are large and are run at 30-50 volts overnight
--this problem happens with all of my Southerns, so I am not looking at a
polymorphism.

Does anyone have any other ideas about what may be causing the problem?

Thanks for your help; I've learned a lot from this group.

Paul



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