Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 15)

8.4 Linear Gradient Gels
For linear gradients, the volume in the mixing chamber (Vm) equals the volume in the
reservoir chamber (Vr). Try volume in each chamber is one-half of the total gel volume (VT):
VT = Vm + Vr or Vm = Vr = 0.5 VT
First calculate the total gel volume required. Then determine the amount needed for the
mixing and reservoir chambers. It is easier to prepare a volume slightly more than the required
gel volume and then quantitatively transfer the required volume into mixing or reservoir
chamber.
Example 1: Describe how to prepare a linear 10-20% gradient for casting six 16 x 20
x 0.15 cm gels in a PROTEAN II multi-casting chamber. The slab gels
will be used for 2-D work and a stacking gel is not required.
1. When a gel casting chamber is used, the gradient must be poured by bottom filling.
Therefore, the mixing chamber of the Model 395 Gradient Former must contain the lighter
(10%) monomer solution and the reservoir chamber must contain the heavier (20%)
monomer solution.
2. When using a multi-gel casting chamber, the total gel volume required depends on sev-
eral factors, including gel thickness, number of gels, gel length, and amount of space-
filling. To precisely determine the volume required for six 16 x 20 x 0.15 cm gels, set up
the casting chamber with he glass plates, spacers and the necessary space fillers and mea-
sure the volume of deionized distilled water required for a 20 cm length. In this example,
let us assume that the total gel volume needed is 310 ml (your actual required gel volume
may differ slightly from this hypothetical value). Disassemble the chamber and rinse and
dry the parts.
3. Reassemble the multi-gel chamber as described in step 2.
4. The amount of monomer required for each chamber will be 155 ml (310/2). It is best to
prepare 170 ml each of a 10% and 20% monomer solution and to quantitatively transfer
155 ml of each solution into their respective chambers. The formulation for each monomer
solution can be calculated using Table 8.2 and a multiplying factor of 1.7 (170 ml required
volume/100 ml volume in Table 8.2).
Monomer Concentration 10% 20%
30% stock 56.6 ml 113.2 ml
Distilled water 68.3 ml 11.7 ml
1.5 M Tris-HCl, pH 8.8 42.5 ml 42.5 ml
10% (w/v) SDS 1.7 ml 1.7 ml
Degas
10% ammonium persulfate 850 µl 850 µl
TEMED 85 µl 42.5 µl
Total monomer volume 170 ml 170 ml
155 ml of the 10% solution will be transferred to the mixing chamber and 155 ml of the
20% solution will be transferred to the reservoir chamber.
5. Set up and cast the gradient as outlined in Section 5, steps 4-8. Overlay the gels with an
equal volume of water.
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