smeared 1st sequencing runs

Richard J. Dudley rdudley at nrc.uab.edu
Thu May 22 13:30:48 EST 1997


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
 
The #1 problem I've seen with poorly performing sequencing gels (either
pouring poorly or running poorly) is lack of elbow grease in the
cleaning step.

I have no affiliation with Gold Biotech, but I do use their sequestrip
and sequesoap and recommend them over chloroform and NaOH soaks. 
They'll give you a free sample.  The Sequestrip does remove Rain-X,
which is good or bad, depending on how you look at it (L always
re-siliconize, so I think it's good).  It takes me about 20 min from the
time I start washing to the time I reassemble my rig.  Here's what I do:

1) Scrape the plates with a razorblade to get rid of chunky stuff, and
wash off.

2) Working one plate at a time, pour a few mls of strip on the plate and
scrub scrub scrub well with kimwipes..  Get a lottle elbow grease into
it.  Rinse off the strip and repeat (this is probably excessive, but I'm
like that with cleaning).

3) Pour some sequesoap onto the plate and scrub.  Rinse and repeat (I've
found two soapings to be important).

4) Shake excess water off and scrub with some 95-100% ethanol.  Scrub
well with kimwipes--change when soaked.  Repeat.

5) Let the plates totally dry, and with a couple of clean, dry kimwipes
between your hand and the plate, feel the plate surface.  Move your hand
all over the plate applying light pressure.  It should glide smoothly. 
Wherever the kipwipes encounter friction, scrub that spot with ethanol.

6) To siliconize a plate, apply two coats of rain-X (or whatever), let
dry a minute or so, and with a damp kimwipe, wipe off the excess.

It may take a try or two to figure this all out (people always look at
me quizically when I try to explain the "feel the plate" idea...), but
you should get very clean plates.

One word of caution:  don't press down really really hard on a plate
that's spanning a sink.  It could be bad.

I hope this helps!

--- --- ---
Richard J. Dudley (rdudley at nrc.uab.edu)                            
Department of Neurobiology                    
University of Alabama School of Medicine       
http://www.nrc.uab.edu/



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