Doublets after electroelution
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,
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