Pouring sequencing gels w/o leaking

Tae Shin tshin at husc8.harvard.edu
Mon Jun 28 22:55:22 EST 1993


	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.  Keep applying the PA at a rate which
matches the PA spreading out between the plates.  Don't worry once it hits
the edges, as surface tension will keep it from spilling out.
7) Believe it or not, the PA will reach the other end of the plates and
fill all the spaces.  Keep an eye out for bubble formation, which can be
nipped in the bud by lightly tapping on the glass.
8) Once the plates are 'filled', wrap the far end (bottom) in cellophane. 
This will keep PA from spilling out too much.
9) Insert the comb.  This will be very easy, since nothing is squeezing
the plates together.
10) Put two more clamps on each side, one near the top and
one near the bottom.  Be sure the clamp is over the spacer.
11) Clamp all across the top edge; ensure that the clamps are squeezing on
the comb.  Some gel may overflow from the bottom edge, but the cellophane
will limit this if it was tightly wrapped.

Enjoy your perfect gel.  No tape, no bubbles.  

T.B. Shin
tshin at husc8.harvard.edu
shin at slsvax.harvard.edu



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