Vertical Agarose gels, help
John Brunstein
brunstei at unixg.ubc.ca
Tue Jun 4 15:55:10 EST 1996
OK :-)) Been there, seen those problems, and solved them! Here's how
I handle them (this is for 1.5% agarose, 1% glycerol gels 1.5mm thick,
about 20x20cm).
(1) Slippage of the gel from between the plates (boy, is it ever a
bummer to see your hard-won bandshift samples slide right out of the
plates and into a sodden little lump an hour into the gel run!) Before
pouring the gel, I run a bit of sequencing tape (the yellow electrical
stuff) across the bottom of the gel just inside the spacer, in a sort
of 'U' shape...(I'll attempt a bad ASCII art rendition below). Leave
these on for the run, as the gel starts to slide it hits them and holds
the gel in place..but since most of the bottom is unobstructed, current
flow is not interfered with
| | | |
| | GEL | |<---- Spacer
| | | |
| |T T|<----- Tape runs down, around bottom, and
| |T T| | partly up far side
-------------------
(2) Removal of comb. Another really frustrating problem...but easily
solved with a bit of practice. The problem is that as you remove the
comb, a vacuum is created in the well and the gel collapses into it,
causing tears. Solution: I use a thin spatula to pry at the glas
plates as I ease the comb out little by little...you'll have to move the
spatula around a bit to ensure that all wells get air into them as you
work the comb out, so don't rush it...but after a few wrecked gels
you'll get the hang of it.
Hope these answers solve your problems, they took me a few weeks of
trial and error to settle on as the final system. I use these gels
routinely for bandshifts (my DNA is too large for acrylamide systems),
and I no longer have any problems with preparing them. There is a bit of
a problem with getting the wells really clean, bits of agarose tend to
stay stick in there despite my best attempts to cut them out, and these
can make the results messier than one might like..but the sytem works.
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