smeared 1st sequencing runs

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Thu May 22 09:09:17 EST 1997


In article <338398d4.0 at hermes.grace.irl.cri.nz>, BulmanS at crop.cri.nz
(BulmanS) wrote:

> Hi there,
> hereabouts, we still do some of our own radioactive sequencing [Thermo 
> Sequenase, internal 33-P, 6% gels], and have recently been having
trouble with 
> our first loadings.  Periodically, these completely smear-out while the
second 
> loadings remain fine.  On some gels, regions of the 1st runs smear out while 
> others remain OK.  The sequencing reactions are fine, since new gels resolve 
> the problem.  I initially thought this was a problem of gel [over]heating but 
> the smearing continues to occur at seemingly random intervals.  Has anyone 
> else had similar problems ?  We're at a loss for an explanation to this 
> irritating but not critical occurance.  Maybe some variation on the heating 
> theme before or during the run, or the silane ..... ??
> cheers then
> 
> Simon
> bulmans at crop.cri.nz

Hi Simon,
The problem might be excess silane on your plates. Try soaking the
precleaned plates in 0.5M NaOH for 60min, then clean with plenty of warm
water and a brush. There is definitely no need for ANY detergent, neither
"normal" soap nor "specialized" stuff as suggested in one post.

Hope this helps,
Frank

-- 
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
D-78434 Konstanz
Germany



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