1. Use distilled water to clean all electrophoresis equipment. Wipe with Kimwipe, and set the components out to dry.
2. Assemble the casting stand.
3. Check assembly for leaks using distilled water. Fill the space between the glass plates; if the unit leaks, reassemble and try again. If not, pour the water out and dry between plates using a folded paper towel, Kimwipe, or filter paper.
4. Mix all resolving gel components together except the 10% APS and TEMED in a 50 mL centrifuge tube. Seal tube and tip back and forth gently to mix.
5. Add 10% APS and TEMED, seal tube and tip back and forth gently to mix.
6. Transfer solution to each of the two plate sandwiches using a 5 or 10 mL pipette. Do not fill all the way! There should be ~1 cm of space between the top of the short plate and the resolving gel level (right in the middle of the green plastic bar behind the glass plates).
It is important that you add the correct amount of gel. Too little and your resolution will be poor; too much, and there won’t be room for a stacking gel on top. Be careful to avoid bubbles, which will inhibit polymerization and distort protein migration. Replace leftover gel in centrifuge tube.
7. Using a pipette or wash bottle, slowly and very gently add just enough 0.1% SDS solution to cover the resolving gel without disturbing it. The SDS solution is there to keep the gel from drying out and to protect it from oxygen, which will inhibit the reaction.
8. Wait 15-30 minutes. Check to see that the leftover gel in the centrifuge tube has polymerized, and if it has, tilt the gel former slowly to confirm that the resolving gel under the SDS solution has polymerized completely.
9. Pour the SDS solution into a Kimwipe, and rinse the top of the gel very gently with dH2O.
10. Mix all stacking gel components together except the 10% APS and TEMED in a 15 mL centrifuge tube.
11. Add 10% APS and TEMED, seal tube and invert several times to mix. Add stacking gel solution to the top of the resolving gel using 5 mL pipette, returning extra solution to centrifuge tube. The stacking gel solution should almost fill the remaining space – leave ~ 2-3 mm between the top of the stacking gel and the top of the short plate. Immediately thereafter, insert the comb. Start from one side and 'brush' air bubbles off to one side.
12. Wait 5-15 minutes. Check to see that leftover gel in the centrifuge tube has polymerized.