Fixing sequencing gels
daj at nhm.ic.ac.uk
Thu Nov 4 02:58:22 EST 1993
Hi Jin and everyone,
In our experience, we get warping when part of the gel comes off and
part stays on the plate. If just a corner comes off, we tend to leave the
rest on the plate but if it looks like a significant portion of the gel is
lifting, we encourage the whole thing to come off with gentle rocking so
it can strech evenly (and then pray it doesn't fall off when we lift the
plates out of the wash solution). We use an aspirator attached to the tap
to suck off most of the wash solution (to below the top of the plate)
before attempting to lift the plate. Some small pieces of glass (or
heavy coins) placed on the gel can help keep it in place when lifting the
plate. Similarly, if you get distortion ridges in the gel once it has been
lifted onto filter paper it is sometimes possible to flood the area with
wash solution and (gently) press the ridges down (you need to use lots of
wash solution as you are effectively trying to float the gel away from the
paper and allow it to shrink back down to size).
Obviously, this is only really safe with sulphur gels. I wouldn't like to
play around with phosphorus gels in this manner but I guess that most
phosphorus users wouldn't bother washing their gels anyway.
To try and prevent lifting in the first place, let the gel/plates
assembly cool for about 15 minutes before trying to separate them and make
sure that everyone in the lab uses silane on only one of the plates (and it
is the same plate for everyone - trying to push a gel back onto a
silanised plate is no joke).
We prewet the filter paper in the wash solution to allow it to stretch
before placing it on the gel to minimise streching artefacts (same
principle as hanging wallpaper). if you do this, you will need to put
tissues on the top surface of the plate/gel/paper assembly to suck out
excess fluid or the gel won't stick to the paper.
We use 10%methanol /10% glacial acetic, lower concentrations also tend to
encourage gel expansion.
Hope this helps - when all else fails, go away and have a cup of coffee and
try again later when you have calmed down. (the probability of loosing the
gel being directly proportional to the importance of the data on it!).
It won't hurt to leave the gel in wash for longer than the usual time
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)
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