Hoefer SE400 - Manual (Page 21)

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2.2.3 Gradient gels
Linear gradient gels can be poured in the gel
caster. For easy gradient mixing, we recom-
mend using one of the Hoefer SG series gradient
makers. Gradient gels are poured from the top
of the caster with a cannula if using the provided
gel caster or from the bottom if using a Hoefer
multiple gel caster (see instructions accompany-
ing the caster). Once the gradient gel polymer-
izes, a stacking gel is poured.
1
Assemble the glass plate assembly into the caster as
described in section 2.1.2.
2
Set up the monomer solution flow path. Run a length
of clear vinyl tubing through a peristaltic pump.
Attach one end of the tubing to the gradient maker
outlet port and the other end to a 20 cm cannula.
(The outside diameter of the cannula must be less
than the spacer thickness.) Place the cannula so
that it rests at the bottom of the sandwich, midway
between the spacers.
3
Prepare the monomer solution. Calculate the total
volume needed. Prepare one half of this volume of
higher and the other half of lower % acrylamide solu-
tion. (
Optional: Add 15% sucrose or 25% glycerol
[final concentration] to the higher % solution to
improve layering.)
4
Pour the “light” solution into the reservoir chamber
(the chamber furthest from the inlet). Open the
stopcock long enough to displace air between the
chambers and then close. Pour the “heavy” solution
into the mixing chamber and place a stirring bar into
this chamber. Place the gradient maker on a magnetic
stirrer and begin stirring at a rate that does not
introduce bubbles in the solution.
Note: With Coomassie ™ Blue,
it is possible to detect 1 µg in
a single band; with the more
sensitive silver stains, it is
possible to detect as little as
10 ng.
Fig 6. Pouring a gradient gel.