DNA stuck in wells
Mr SMNN Faruque
sfaruque at uk.ac.mrc.hgmp
Tue Jan 10 06:41:10 EST 1995
> > 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.
> It might be due to incomplete dissolution following an ethanol
> precipitate. Drying in a speed-vac can make pellets very hard to
> dissolve. You could try heating to 60 degrees for 15 minutes and
> vortexing a few times.
The DNA has not been precipitated since labelling, just put thru' a G50
column. Any undissolved DNA prese3nt should be cold, shouldn't it?
> Since the DNA in buffer looks the same, I suggest changing buffer conditions.
> The DNA in buffer should give a nice tight band on the gel.
I've tried glycerol loading dye (a la Maniatis) and both the protein-DNA
binding buffers mentioned above BCA and Scholer. (NB Scholer, with an
umlaut over the o, is 10mM Hepes pH7.8, 1mM spermidine, 5mM MgCl2, 50mM
Kcl, 9% glycerol).
I noticed that I'd been using 19:1 acrylamide:bis acryl instead of the
recommended 29:1, although I've seen references to using that, I tried
29:1 and found only 1/2 of the probe stuck in the wells. That's still too
much tho', isn't it?
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