Preparing Vertical Discontinuous Buffer Sds Polyacrylamide Gels

Assembling of the gel moulding cassette. Clean the two glass plates (size: e.g. 10 cm x 8 cm), the polytetrafluoroethylene comb, the two spacers and the silicone rubber tubing (diameter e.g. 0.6 mm x 35 cm) with mild detergent and rinse extensively with water. Dry all the items with a paper towel or tissue. Lubricate the spacers and the tubing with non-silicone grease. Apply the spacers along each of the two short sides of the glass plate 2 mm away from the edges and 2 mm away from the long side corresponding to the bottom of the gel. Begin to lay the tubing on the glass plate by using one spacer as a guide. Carefully twist the tubing at the bottom of the spacer and follow the long side of the glass plate. While holding the tubing with one finger along the long side twist again the tubing and lay it on the second short side of the glass plate, using the spacer as a guide. Place the second glass plate in perfect alignment and hold the mould together by hand pressure. Apply two clamps on each of the two short sides of the mould. Carefully apply four clamps on the longer side of the gel mould thus forming the bottom of the gel mould. Verify that the tubing is running along the edge of the glass plates and has not been extruded while placing the clamps. The gel mould is now ready for pouring the gel.

Preparation of the gel. In a discontinuous buffer SDS polyacrylamide gel, it is recommended to pour the resolving gel, let the gel set, and then pour the stacking gel since the composition of the two gels in acrylamide-bisacrylamide, buffer and pH are different.

Preparation of the resolving gel. In a conical flask, prepare the appropriate volume of solution containing the desired concentration of acrylamide for the resolving gel, using the values given in Table 2.2.31.-1. Mix the components in the order shown. Where appropriate, before adding the ammonium persulphate solution and the tetramethylethylenediamine (TEMED), filter the solution if necessary under vacuum through a cellulose acetate membrane (pore diameter 0.45 pm); keep the solution under vacuum by swirling the filtration unit until no more bubbles are formed in the solution. Add appropriate amounts of ammonium persulphate solution and TEMED as indicated in Table 2.2.31.-1, swirl and pour immediately into the gap between the two glass plates of the mould. Leave sufficient space for the stacking gel (the length of the teeth of the comb plus 1 cm). Using a tapered glass pipette, carefully overlay the solution with water-saturated isobutanol. Leave the gel in a vertical position at room temperature to allow polymerisation.

Preparation of the stacking gel. After polymerisation is complete (about 30 min), pour off the isobutanol and wash the top of the gel several times with water to remove the isobutanol overlay and any unpolymerised acrylamide. Drain as much fluid as possible from the top of the gel, and then remove any remaining water with the edge of a paper towel.

In a conical flask, prepare the appropriate volume of solution containing the desired concentration of acrylamide, using the values given in Table 2.2.31.-2. Mix the components in the order shown. Where appropriate, before adding the ammonium persulphate solution and the TEMED, filter the solution if necessary under vacuum through a cellulose acetate membrane (pore diameter: 0.45 pm); keep the solution under vacuum by swirling the filtration unit until no more bubbles are formed in the solution. Add appropriate amounts of ammonium persulphate solution and TEMED as indicated in Table 2.2.31.-2, swirl and pour immediately into the gap between the two glass plates of the mould directly onto the surface of the polymerised resolving gel. Immediately insert a clean polytetrafluoroethylene comb into the stacking gel solution, being careful to avoid trapping air bubbles. Add more stacking gel solution to fill the spaces of the comb completely. Leave the gel in a vertical position and allow to polymerise at room temperature.

Table 2.2.31.-1. - Preparation of resolving gel

Solution components

Component volumes (ml) per gel mould volume of

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