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Hoefer DALT System Casting Gradient Gels
Amersham Biosciences
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2. Configure the gradient divider for the number of gels you are casting. If necessary,
calibrate the gradient pump flow rate. See “Calibrating the Peristaltic Pump” on
page 17.
3. Be sure that the faceplate screws on the gradient maker are tightened hand tight and
the gradient-maker lines are all clamped off. There are three clamps: one coming
from each chamber and one after the bow-tie mixer. Close all three.
4. Prepare a sufficient volume of gel overlay solution (water-saturated n -butanol). You
need 0.75 ml of overlay for each cassette, or about 20 ml for a set of 25 cassettes.
5. Make up 200 ml of displacing solution (page 42).
6. Make up the gel acrylamide solutions from the stock mixes, but do not add the
10% ammonium persulfate (APS) and 10% N,N,N',N',-tetramethylethylene-
diamine (TEMED). See “Gradient Gel Solutions” on page 49.
Pouring Gel Solutions for Gradient Gels
1. Prepare the gel caster, as described on page 12, placing gel labels in each cassette.
2. When you are ready to cast the gels, add the APS and TEMED and mix each gel
solution thoroughly. Vary the amount of TEMED added to control the rate of
polymerization.
Once these reagents are added, polymerization begins. You have about 10 minutes
to cast the gradient before the gels begin to solidify at the top. Work rapidly and
carefully.
3. Pour the light solution into the right side of the gradient maker (the chamber that is
wider at the top—“Light in Right”).
4. Fill the tubing between the light and heavy chambers with light solution.
Carefully open the clamp on the light chamber exit tube (a) and then very slowly
open the heavy chamber exit tube clamp (b). Allow light solution to fill the tube
coming from the light chamber all the way to the “Y” connector and back up to the
point at which the heavy tube enters the heavy chamber. Fill the entire tube with
LIGHT solution (no bubbles), but do not allow light solution into the heavy
chamber itself (Figure 11).