Part A: Assemble the gel apparatus, using spacers to hold glass plates apart. Clamp and mount in holder. Make sure gel is mounted firmly by tightening the cams.

Part B: Make a separating gel, but do not add TEMED. Mix gently, then add the TEMED and mix again. IMMEDIATELY pipette into gel apparatus, stopping about 2-2.5 cm from the top. You can use an autopipetter to speed this step up. Overlay gently with 1-2 mL butanol.

Part C: Make a stacking gel, but do not add TEMED. Mix gently. Pour butanol off the separating gel once it has polymerised and rinse with 1-2 mL of the unpolymerised stacking gel mixture and pour this off. Add the TEMED to the stacking gel mixture, mix well, and IMMEDIATELY pipette on top of the separating gel (again using an autopipetter, if needed). Quickly insert the comb to a depth of 1.2-1.5 cm. Prepare your samples.

Part D: When you samples are ready and the stacking gel is polymerised, pour some of the running buffer (contains SDS) over the comb to help loosen it. Slowly pull the comb upward using a side-to-side motion. Fill all the slots with running buffer and straighten the slots with a spatula, if necessary.

Part E: Note that we use a special pipette tip that is longer and thinner than normal pipette tips. Load the gel.

Part F: As soon as the samples are layered, clamp the top buffer compartment onto the gel apparatus and remove the bottom compartment (use the cams from the bottom one to tighten the top one). Flow water through the tubing in the buffer chamber to keep the gel cool during electrophoresis. Fill the buffer chamber with about 4L of running buffer and put the clamped gels into the chamber. Gently put about 500 mL of buffer in the top compartment.

Part G: Connect the power supply and then apply about 20mA per gel, increasing to about 30mA per gel and run for about 4 hours (will be finished when the blue tracking dye reaches the bottom of the gel).

Part H: The electrophoresis is stopped and the buffer in the upper chamber is discarded. The gels are removed and unclamped. The glass plates are gently separated and the spacers are removed. The gel is gently removed from the glass and put into a tray (note there are two different methods to do so). The second way of tipping the plate upside down is much safer; you can rip your gel by pulling it off the glass if you are not careful. The gel is usually fixed and stained (the staining procedure used changes depending on your experiment). Gels are agitated continuously throughout the fixation and staining steps. The gel is destained and photographed to make a permanent record.