Doublets after electroelution

David Glover D.J.Glover at bham.ac.uk
Wed May 31 16:19:59 EST 1995


                      I am trying to purify three 256bp DNA fragments for use 
as probes in Northern blot analysis. One of the fragments is cut from a 
plasmid and the other two are prepared by PCR from genomic DNA. When I run the 
digest or PCR products on a 4% acrylamide gel I get nice single bands of the 
correct size. However, when I cut out the band and electroelute the DNA I end 
up with two bands when checked on a gel. One band (lower) is the correct size 
and the other (upper) is about twice the size of the lower band.

                     This is complicated further by what appears to be a 
random factor. Some elutions (about 1 in 10) work fine yielding a single band 
of the correct size. These preparations work when radiolabelled and used in 
the Northerns.Preparations with the extra band label ok (as checked by running 
labelled prep. on a gel and autoraiography - both bands label) but do not work 
in the Northerns.

                     Does anybody have any idea where this extra band is 
coming from? If the two bands are related, then why don't they work in the 
Northern blots? I've tried all sorts of variations on my method: long/short 
elution times, high/low current, no. phenol/chloroform extractions, eluting at 
4 oC, agarose/acrylamide gels, loading varying amounts on the gel etc. When I 
run the doublets on an acrylamide gel  a second time and cut out the lower 
band for electroelution I still end up with a doublet again!

                    Thinking the problem was the electroelution I tried 
crushing the gel slice and eluting at 37oC O/N in 0.5M ammonium acetate, 1mM 
EDTA as described in Maniatis.To my amazement I still got the two bands!

                    My basic method is:
1) run digests/PCR reactions on 4 % acrylamide-TBE gel at 8 V/cm with cooling 
    ca. 3 hours
2) cut out band and electroelute against 0.2x TBE, 20 mA 30 min.
3) extract eluate once with phenol/chloroform, once with chloroform
4) EtOH precipitate -20 O/N (with 0.1 vol 3M Na Ac).

                   I would be most grateful if anyone could explain what's 
going on and how to solve the problem. It's driving me mad!

Thanks in advance for any help,
Dave Glover.



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