DNA stuck in wells
maga at vetbio.unizh.ch
Tue Jan 10 08:52:04 EST 1995
In article <sfaruque-0701951437100001 at cfd-lab.umds.ac.uk>,
sfaruque at uk.ac.mrc.hgmp (Mr SMNN Faruque) wrote:
> Why, on occasions, does DNA seem to stick in wells?
> I've seen it before in horizontal gels, sequencing gels and now I've had
> it in a vertical non-denaturing gel.
> I've tried the 4% non-deturing gel (a band-shift assay) with TBE and TAE,
> with Scholler buffer and BCA and the probe (144bp) doesn't seem to move
> any distance into the gel even in the lane with just buffer and probe.
> Current Protocols... has no mention of this problem in its troubleshooting
> section and I'm not sure what to try next.
> Nadeem Faruque
I performed bandshifts with 8% polyacrylamide gel for 4h at 250 V in 90 mM
Tris-borate buffer, 10 mM EDTA pH 8.0. The probe was indeed smaller than
yours, it was a 60mer with a 15mer primer annealed. The protein bound was
200 kD. Anyway, under the same conditions I succeded also in bandshifting a
400mer probe with multiple 15mer primers annealed. I don't know which
problem are you suffering. Are you sure that your probe is not contaminated
by proteins? I would suggest to prepare your probe (before incubate it with
your protein of course) by digestion with Proteinase K and then extraction
with phenol, since ethanol extraction can cause also proteins to
precipitate. In any case, you will always have some radioactivity in the
wells. Another problem could be overloading of the gel. Have you tried
different amounts of probe?
G. Maga, PhD
Institute of Biochemistry UNI-Irchel Zuerich (CH)
e-mail: maga at vetbio.unizh.ch
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