Pouring sequencing gels w/o leaking
Mark S. Whitsitt, N5RJF
whitsitt at bioch.tamu.edu
Wed Jun 30 09:24:00 EST 1993
In article <tshin.741326122 at husc.harvard.edu>, tshin at husc8.harvard.edu (Tae Shin) writes...
> The method I've used is fast and nearly fool-proof. Almost anyone
>can do it the first time. The reference is from Biotechniques (1991),
>#12, p78. Here's the method I use with an almost 100% success rate.
>1) Wash both plates with detergent and water. Let drip dry, then wipe
>clean with 200pf EtOH.
>2) Silanize _both_ plates. Even if you use a permanent/long-term
>silanizing agent, it's a good idea to polish it once with SigmaCote.
>3) Lay the larger plate on a flat surface or stand (e.g. a pipet tip box).
>Place the spacers on each side, then lay the shorter/notched plate on top.
>Make sure the plates and spacers are aligned. Check with a bubble-level
>that the plates are flat.
>4) Put _one_ clamp on each side, at about the middle. Position it so that
>it is clamped on the spacer. This is not sealing anything, but it just
>keeps things from moving around.
>5) Mix/degas your sequencing mix. Add and APS and TEMED, and keep on ice
>to slow down the polymerization.
>6) Use a 25-50mL pipet to draw up some PA, then slowly apply it to the top
>of the plates (where the comb will be), in the middle. Capillary action will
>pull in the PA between the plates.
>Enjoy your perfect gel. No tape, no bubbles.
Just a note... you will be able to see bubbles begin to form early on as a
portion of the PA soln will not wet the glass plates... tapping the plates over
this area allows the PA to wet the plates. Also, I only silanize the larger
plate and leave the other one unsilanized. It does not hurt the effectiveness
of the procedure and allows you to separate the plates (I think) more easily as
the gel has a much greater affinity for the unsilanized plate and it won't want
to stick to both plates.
Also, every once in a while, I use a bit of AJAX cleanser on the plates,
scrubbing gently with a Kim wipe. I then re-silanize the large plate. I do
this when bubble formation begins to become a big problem. I haven't scratched
my plates yet...
I started using this method when I first saw it in Biotechniques and have never
gone back to tape. This method is a dream! Others in the lab saw me doing this
and now *they* will never use tape again to pour a sequencing gel!
Mark S. Whitsitt, N5RJF Texas A&M University, Dept of Biochemistry
Internet: mwhitsitt at tamu.edu College Station, Tx. 77843-2128
AMPRnet: n5rjf at n5rjf.ampr.org (409) 845-0832
Packet/APLINK: N5RJF @ KE5HE.TX.USA.NA
More information about the Methods