Need help - black dots on DNA sequencing gels?
tan at aeolus.vmsmail.ethz.ch
Tue Sep 6 12:26:36 EST 1994
In article <34hulm$q6 at jhunix.hcf.jhu.edu>, reminto at jhunix.hcf.jhu.edu
(Robert E Minto) wrote:
> I am in need of some help removing interfering black dots from my
> polyacrylamide sequencing gels.
>The gels contain black dots which are
> concentrated at the top of the sequencing gel and are ONLY in the lanes.
> They actually interupt the lanes, suggesting particles in the gel. The
> gels even contain dots when poured from fresh reagents (Mol. Biol.
> grade), with aspirator-degassed solutions carefully swirled with TMED and
> APS to avoid bubbles, and polymerized for 2 hours before using
> immediately. Both vertical and horizontal (capillary action) pouring
> techniques give the same result. The buffer is 1 x TBE.
Yeah, I get these black dots in my sequencing gels as well. There are
typically 0.1 to 0.3 mm in diameter and streak downwards. I agree with
your assessment that they are particles in the gel. Most likely candidate
for the source of the particles is the TBE buffer, which we all know can
precipitate in its 10x stock. In particular, if you pour 10xTBE buffer
from its bottle that is normally capped, you're bound to wash off some
particles from the rim of the bottle.
I usually don't worry about these dots in my sequencing gels as they don't
affect my ability to read the sequence. But they can be annoying when you
have a nice footprinting gel or some other gel that you want to publish.
My solution in those cases is to filter the gel mix through 0.44 micron
filters just prior to adding the TEMED and ammonium persulfate. That
usually eliminates the problem.
I don't have a good explanation for why the dots appear mainly at the top
of the sequencing gel (I just looked at some of my sequencing gels and see
the same thing: most dots are in the top third of the gel, generally no
dots in the bottom two-thirds of the gel). But I'm pretty sure I know why
the dots are only in the lanes. I was confused by this observation until
I looked at my gels and saw the same phenomenon there. The dots are black
because the radioactive DNA is getting hung up on the particles. But
there isn't any radioactivity in between the lanes. So although there
are probably particles throughout the (top part) of the gel, you only see
them when they occur in a lane.
Anyone with an idea for why the dots appear almost exclusively in the top
half or third of the gel?
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: tan at aeolus.vmsmail.ethz.ch
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