sequencing gel won't stick to Whatman paper

Brad Nicholson brad_nicholson at hlthsci.med.utah.edu
Fri Jun 14 08:16:43 EST 1996


In article <v01530500ade51e3a6d3d@[142.20.8.21]>, jlight at SICKKIDS.ON.CA wrote:

> After I run a sequencing gel (using 35S-dATP for labelling) I have a lot of
> problems getting the gel to stick to Whatman paper.  
>Gel transfer problems snipped<

Hello what we do in our lab is to start with two sheets of Whatman paper
that are slighly larger than the gel.  After seperating the plates I lay
the plate carrying the gel on a diaper or covering of paper towels.  Then
I lay one of the Whatman sheets on top of the gel.  I hold the paper along
the long axis with the sides curved up so that the center is hanging down
in a U shape.  Then I gently lower the filter paper so that it touches in
the middle of the gel, followed by slowly lowering the edges so the gel
makes contact with the filter from the center out towards the edges.  Then
I gently the Whatman paper to make sure that everything is in contact with
the gel.  The next step is to flood the surface of the filter with water,
I use a squeeze bottle or two and make sure that the entire surface is
soaked.  Then I use a 10 ml pipette to roll out any excess liquid and to
make sure that the gel is adhering to the paper.  Then I lay a dry sheet
of Whatman on top of the soaked piece/gel.  The pipette is used again to
gently roll over the dry sheet so that is contacts the soaked sheet/gel. 
Then I gently flip the entire assembly over onto the diaper/paper towels
(This isn't as bad as it sounds, you just hold the ends of the sequence
plate to prevent the paper from flopping around and carefully pick it up
and turn it over.  If you are really paranoid you can cover the plate with
a spare sequence plate to make sure nothing shifts during the rotation.) 
To separate the gel you start at the thickest end (or the end without the
wells) and slowly lift up on the glass plate.  The gel/filter paper should
fall away with gentle coaxing and gravity.  Don't lift the glass plate up
to much as it is possible to tear the gel.  If you run into a sticky spot,
use a squeeze bottle of water and shoot the water up between the gel and
the glass plate and lower or tilt the plate so the gel can release from
another angle.  

Wow, this got long.  This technique seems to work pretty well when
combined with really clean plates, we hardly ever get sticky spots or
tears.  

Good luck,

Brad
=====================================================================
Brad Nicholson                      |"If it worked the first time, 
Department of Pathology             | it wouldn't be research.'
University of Utah                  | ...Brad Nicholson
Salt Lake City, UT 84132            | My opinions are solely my own.
brad_nicholson at hlthsci.med.utah.edu |
or: (801)-581-4365                  | iligitimi non corborundrum
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