How to perform polyacrylamide gel electrophoresis in the lab

The Department of Biological Sciences at the University of Alberta brings you this video tutorial specifically relevant to your student laboratory courses, specifically microbiology. If you're a student at any school of biology, this information will be helpful for learning how to perform polyacrylamide gel electrophoresis in the lab.

Part A: Assemble the gel apparatus.

Part B: Make a separating gel, but don't 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. 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 polymerized and rinse with 1-2 mL of the unpolymerized 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. Quickly insert the comb to a depth of 1.2-1.5 cm. Prepare your samples.

Part D: Pour some of the running buffer 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.

Part E: Load the gel.

Part F: Clamp the top buffer compartment onto gel apparatus and remove the bottom compartment. Flow water through the tubing in the buffer chamber. 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.

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. Gels are agitated continuously throughout the fixation and staining steps. The gel is destained and photographed to make a per