black dots on DNA sequencing gels?

Susan E Ingraham singraha at magnus.acs.ohio-state.edu
Tue Sep 6 10:35:11 EST 1994


In article <34hulm$q6 at jhunix.hcf.jhu.edu>,
Robert E Minto <reminto at jhunix.hcf.jhu.edu> wrote:
>I am in need of some help removing interfering black dots from my
>polyacrylamide sequencing gels.  The gels are prepared by the U.S.
>Biochemical Sequencing support guide protocol which contains 8M urea (and
>appears to be identical to Maniatis, 6% acrylamide, 19:1
>acrylamide:bisacrylamide).  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.  The operating
>temperature is ca 40-45 degrees at 60W power.
>
You don't mention having filtered the acrylamide solution; we do so routinely 
(right before degassing) and don't seem to have this problem.  This also seems 
to indicate that you have some sort of particulate matter in your gel matrix.

-Sue I.
 singraha at magnus.acs.ohio-state.edu
 Research Assistant 2, Neurogenetics Laboratory



More information about the Methods mailing list