Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 16)

8.5 Exponential Gradient Gels
For an exponential gradient gel, the volume in the mixing chamber is one fourth the total
required gel volume (0.25 VT) and the volume in the reservoir chamber equals the total
required gel volume (1.0 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
chambers.
Example 2: How to prepare a concave 5-15% exponential gradient for one 16 x 16 x
0.15 cm gel with a 2 cm stacking gel to be cast in the PROTEAN II xi casting stand.
1. A concave exponential gradient must be cast from the top. The heavier 15% monomer will
be placed in the mixing chamber and the lighter 5% monomer will be placed in the reser-
voir chamber of the Model 385 Gradient Former.
2. From Table 8.1, 38.4 ml total gel volume is required. 9.6 ml (0.25 x 38.4 or 0.25 VT) of
the 15% monomer solution is required for the mixing chamber and 38.4 ml (VT) of the
5% monomer solution is required for the reservoir chamber. It is convenient to prepare 15
ml of the 15% solution and 50 ml of the 5% solution and quantitatively transfer the
required volumes into the respective chamber.
3. The multiplying factor for the 15% monomer solution is 0.15 (15/100) and the multiply-
ing factor for the 5% monomer solution is 0.5 (50/100). Prepare the gel solution using
Table 8.2:
Monomer Concentration 5% 15%
30% stock 8.3 ml 7.5 ml
Distilled water 28.4 ml 3.5 ml
1.5 M Tris-HCl, pH 8.8 12.5 ml 3.8 ml
10% (w/v) SDS .5 ml 50 µl
Degas
10% ammonium persulfate 250 µl 75 µl
TEMED 25 µl 7.5 µl
Total monomer volume 50 ml 15 ml
9.6 ml of the 15% solution will be transferred to the mixing chamber and 38.4 ml of the
5% solution will be transferred to the reservoir chamber.
4. Set up and pour the gradient as outlined in Section 3.1, steps 2-8. Overlay the gels with
water saturated isobutanol. After the separating gel has polymerized, remove the water sat-
urated isobutanol and wash the top of the gel with distilled deionized water. Prepare the
stacking gel as described in Table 8.2. Cast the stacking gel and insert the comb to form
the wells.
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