Electrophoresis Unit for sequencing
jkropp at itsa.ucsf.edu
Wed Jun 25 19:03:39 EST 1997
In article <33AE9471.190E at worf.molbiol.ox.ac.uk>,
mysun at worf.molbiol.ox.ac.uk wrote:
> Anybody out there has suggestions to buy an Elactophoresis unit
> for manul sequencing. We use Model S2 unit from Life Technologies Inc
> at moment and everyone hates the leakage produced.
> Your input would be highly appreciated.
> B GAO, PhD
Maybe I can save you some money. Try keeping your current unit and pouring
your gels horizontally. Surface tension will keep the acrylamide in the
plates and you won't have gravity attempting to pull it out--causing
leaks. I've been doing it this way for years. Here's how:
The most important thing (as always) is very clean plates. I wash mine
with powdered detergent and cerium oxide (more on that later), scrubbing
like hell with a "Scotchbrite" pad. Cerium oxide is the magic ingredient.
I get mine from Crystalite Corp., Marina del Rey, Cailf., Prod.# 41-271.
If you are unable to access them from the UK, try to find a supplier of
the stuff that is "Polishing Grade". I'd guess that an artisan that works
with glass would know. It is simply a very fine abrasive. And it's cheap.
After plates are clean and dry, treat one with Rain-X (a windscreen
treatment sold at auto parts stores), or silane, etc. Rain-X is lots
Don't seal the bottom of the plates, or the sides. Just put a spacer on
each side and clamp a bit. Elevate the top at about 15deg. from the
horizontal (not critical).
Prep your acrylamide. Don't bother to degas, just use plenty of APS. I use
60ul of 25% for 30ml of gel and 30ul TEMED, and work fast if it's a warm
day. Using a syringe, inject the acryl. soln, drawing the syringe across
the top of the plates so it is not all injected at the same point.
If the gel moves down the plates as a "front" without a lot of "fingers"
you have clean plates. If you get fingers, you're going to get bubbles due
to dirt on the plates. Keep injecting gel, the whole process takes
~2-3min, until a little drips out the bottom. Return the plates to
horizontal, insert comb, and allow to polymerize at least 45min.
If you pour v. wide gels, the top plate will sag a tiny bit, resulting in
failure. Prevent this by inserting a piece or two of spacer in the top,
where the comb will go after pouring.
Everyone I have talked into trying the method likes it. Can't remember who
I stole it from, but I sure didn't invent it. And it won't work without
clean, clean, clean plates. I get one failure per ~25 gels.
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