Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 7)

6. Quickly turn on the stirring motor (to a speed which gives good mixing without foaming),
open the valve stem and the stopcock, and start the peristaltic pump (see Note on the
speed of the peristaltic pump).
7. As the pump removes acrylamide from the mixing chamber, the reservoir solution will be
drawn into the mixing chamber and the gradient will be formed.
8. When the acrylamide solution reaches the desired level in the gel sandwich, stop the
pump, remove the tubing from the gel sandwich, and transfer it to a waste receptacle.
Overlay the gel with water or water-saturated isobutanol. For a continuous system, add the
comb to form the wells.
9. Remove the exponential piston from the mixing chamber and restart the pump to remove
any remaining acrylamide. When the chambers are empty, add rinse water to both the mix-
ing and reservoir chambers and flush out the system as described in Section 2.2, step 12.
3.2 Convex Exponential Gradient Gels
Convex gradient gels are formed by delivering the acrylamide from the bottom, with the
small volume of light solution in the mixing chamber and the large volume of heavy solution
in the reservoir chamber (see Figure 7.4).
1. Calculate the volumes of light and heavy solutions as in Section 3.1, except that the light
solution will be 1/4 the total gel volume and the heavy solution will be equal to the total
gel volume.
2. Set up the equipment as described in Section 2.3, with the needle entering the gel sand-
wich from the bottom through the casting stand gasket. For a continuous gradient, insert
the comb into the sandwich at an angle.
3. Mix and degas the small volume of light acrylamide solution and large volume of heavy
acrylamide solution.
4. Add the initiators to the light solution, swirl 8 to 10 times, and pour it into the mixing
chamber, leaving the valve stem closed.
5. Immediately fix the volume with the piston as described in Section 3.1, step 5.
6. Add the initiators to the heavy solution, swirl 8 to 10 times, and pour into the reservoir.
7. Cast the gradient as described in Section 3,1, steps 6-8.
8. Rinse out the system as described in Section 3.1, step 9.
Section 4
Pouring Two Gradient Gels
Double the volumes of heavy and light solution, and use the Y-connector provided. The
procedures are the same as above. It is essential that the tubing from the Y to the two gels be
the same length, and that the rate of flow be the same from each needle. We advise that this
be checked with water and two measuring cylinders ahead of time.
Note: It is difficult to attain reproducible gradients when pouring two gradient gels.
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