black dots on DNA sequencing gels?

szcooley at chip.ucdavis.edu szcooley at chip.ucdavis.edu
Tue Sep 6 11:19:24 EST 1994


Susan E Ingraham (singraha at magnus.acs.ohio-state.edu) wrote:
: 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


I experienced the same dots even after filtering through Whatman paper. 
Is Whatman paper sufficient?


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