Sequencing gel combs (leakage)

Michael G. Tencza tencza at med.unc.edu
Tue Mar 29 09:42:06 EST 1994


>Hello comrads,

>Has anyone figured out why sequencing gels will leak on many occasions
>allowing loaded samples to flow into adjacent spaces ? I use a "shark's
>tooth" comb and more than half of the time find movemnet of the laoded
>sample between the plate and the comb.

>I often allow my gels to polymerize merely several hours while others
>swear that overnight is necessary. This may lead to a soft edge at the
>top perhaps. Otherwize swelling and seperation of the gel from the
>plate may be occurring during the pre-run.

>Any tips greatly appreciated. At this point I would just a soon load
>into regular analytical wells due to this problem.


>thanks,

>Jim

I am relatively new to the sequencing business and had the same problem
with leakage recently.  The first recommendation I can offer is to make sure
the best combination of long plate to bottom plate is achieved, thus
optimizing the spacing between plates where the comb fits in.  The next thing
is to make sure your comb is replaced in the same orientation between the
plates when you remove the flat side and replace the tooth side (ie: don't 
flip the comb right to left).  The last and possibly most important tip is
to place the top clamps on the glass directly over the comb and not the side
spacers during polymerization of your gel.  This normally insures a good
seal and little or no leakage will occur between lanes.

I have followed these guidelines with two different sets of plates from
two different electrophoresis set ups and have been successful both times.
Note though that it is possible that you have a set of warped plates.  If
this is the case then you need to use a new set.

Good luck,

Mike



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