Procedure for Casting the Gel

The glass plate sandwich of the electrophoresis apparatus is assembled using two clean glass plates and there 1 mm acrylic spacers. The sandwich is locked in the positioned and then sealed along the three sides using molten 1% agar. The separating gel solution is prepared by mixing the reagents in correct proportions. Care should be taken that the freshly prepared APS and TEMED are added last, as they are responsible for the polymerization. Acrylamide being neurotoxic should be handled with care. The solution is mixed thoroughly and filled into the sandwich to the required height. A small amount of carbon tetrachloride is poured on top in order to level the top layer also to cut off oxygen supply. This arrangement is left for 15 minutes, so that the gel solidifies. The top layer of carbon tetrachloride is then poured off and the surface is washed with distilled water.

Now the staking gel solution is prepared by mixing the reagents in correct proportions. On pouring into the plates Teflon comb is slowly inserted into the plates so that no air bubbles get trapped between the two gels. this arrangement is also left for 15 minutes. After the gel solidifies the comb is removed and the wells are washed thoroughly with the tank buffer in order to remove any unpolymerized acrylamide. The sandwich is fitted onto the vertical slab unit after removing the bottom spacer. Care should be taken to see that there are air bubbles between the bottom of the sandwich and the tank buffer, which is filled in the tank of the vertical slab unit.