salt conc. and DNA mobility question

Jody K. Hirsh jkh141 at nwu.edu
Mon Jul 22 10:03:21 EST 1996


Robot Boy wrote:
> 
> Hi all,
> 
> I was wondering if someone out there could help me out.
> 
> I was doing some genomic southerns and performed the restriction digests on
> large volumes...then evaporated some of the volume on a speedvac to drop the
> volume for gel loading. Should I have precipitated the DNA with
> ethanol instead ?
> 
> Two expected bands were slightly shifted up and I don't know if this is a
> result of excess salt from the buffer which affected mobility. I also
> noticed as I was running the gel, that the BPB and Xylene Cyanol migrated
> a little slower for some of the samples. Is this all due to all the
> salt from the buffer ?  On a 100 microliter digest, 10 microliters were
> buffer, and after digestion I then evaporated back down to 15 microliters.
> 
> Any thoughts ?
> 
> thanks for your help,
> 
> Ed Taboada
> Dept. of Biology
> University of Ottawa
> Ottawa, CanadaYes, the salt does shift the relative mobility. The current is greater 
with more salt ions. Alternatively to ethanol precipitation, you could 
add the same amount of salt/buffer to the molecular weight markers.  If 
you have used different buffers for the digests, you could make separate 
lanes of markers for the different buffers. You could calculate how much 
the samples were concentrated and also add the correct amount of 
10x concentrated restriction buffer. (Not a plug for Pharmacia, but they 
have a universal buffer, "one phor all" that might be easier to use).
-- 

Jody K. Hirsh
Northwestern University, Chicago, IL.   USA
jkh141 at nwu.edu



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