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Casting Gradient Gels Hoefer DALT System
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Amersham Biosciences
Casting Gradient Gels
The gradient maker is a simple unit with two chambers, defined by a silicone rubber
gasket clamped between two acrylic plates. The chambers are separated by a movable
divider, which you can modify to define the shape of the gel gradient.
Solutions flow out of the two chambers, join at the Y-connector and then thoroughly
mix in a “bow-tie” in-line pipe mixer that has no moving parts.
Three pinch clamps control the flow at the exits from the light (a) and heavy (b)
chambers, and after the mixer (c) to control flow into the peristaltic pump (Figure 10).
Figure 10. The pump-
assisted gradient maker
A gradient gel results from using two gel solutions of different acrylamide
concentrations and densities, a light solution and a heavy solution. The heavy gel
solution contains glycerol. During the gradient pouring procedure, the mixing ratio of
heavy solution to light solution gradually increases, with the heavier solution
underlaying the light solution. This generates a downward gradient of increasing gel
percentage. To assure balanced flow, when the gradient maker is filled with equal
volumes on each side of the divider, the height of the heavy gel solution in the gradient
maker should be 1 – 2 cm less than the height of the light solution. Under these
conditions, the two solutions are in hydrostatic equilibrium. See “Configuring the
Gradient Divider” on page 16.
Gradient Casting Setup
WARNING Acrylamide is a neurotoxin. Always use mechanical pipettes and wear
protective gloves when working with acrylamide solutions, IPG strips
or surfaces that come in contact with acrylamide solutions.
1. Be sure the entire gel casting system is clean, dry, and free from any polymerized
acrylamide.
Clamp a
Clamp b
Clamp c
Gradient divider
Light chamber
Heavy chamber
Pump