3.15. Handcasting Polyacrylamide Gels

  1. Calculate the amount of 30% acrylamide and diH2O needed for desired resolving gel percentage using Table 1.
  2. Prepare resolving and stacking gels without APS or TEMED.
  3. Prepare 10% APS solution. Use 10 μl for every 1 mg of APS. Note: APS slowly degrades in solution and therefore should be made fresh. Do not use old solutions of APS.
  4. Assemble glass cassette sandwich, and place it in the clamp with a foam strip underneath to prevent the cassette from leaking.
  5. Place the comb into the assembled sandwich. With a marker, draw a mark on the glass 1 cm below the teeth of the comb.
  6. Measure 5 ml of resolving gel into a centrifuge tube.
  7. Add 2.5 μl of TEMED and 25 μl of 10% APS to the tube.
  8. Cap the tube and gently mix the solution by inverting the test tube.
  9. Fill the glass cassette to the mark made in step 5 with resolving gel using a pippette.
  10. Add about 100 μl of isobutanol to the glass cassette. Make sure that the top layer of the resolving gel is completely covered with the isobutanol.
  11. Allow the gel to polymerize for 45-60 minutes.
  12. Remove all of the isobutanol by inserting a piece of filter paper or a Kimwipe.
  13. Place the comb into the glass cassette. Raise one end of the comb until there is enough room between the comb and the glass for a pipette tip to fit between the comb and the glass.
  14. Measure 5 ml of stacking gel into a centrifuge tube.
  15. Add 5 μl of TEMED and 25 μl of 10% APS to the tube.
  16. Cap and gently mix the solution by inverting the test tube.
  17. Slowly fill the remainder of the glass cassette with the stacking gel with a pipette.
  18. Press the comb into the glass cassette. Make sure that there are no bubbles in the gel.
  19. Allow the gel to polymerize for 30-45 minutes.
  20. Remove the comb by pulling it up slowly and gently.

This protocol is revised from the following Bio-Rad guide:

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin 6201.pdf

This video provides an example of how to cast a gel (see below):

https://www.youtube.com/watch?v=EDi_n_0NiF4

Note: The video does not fill the stacking gel as described in the procedure.

Table 1. Polyacrylamide Gel Recipe
Percent

Stacking Gel

(4%)

Resolving Gel
(X%)
30% Acrylamide 1.98 ml 0.5 × X ml
0.5M Tris-HCl, pH 6.8 3.78 ml -
1.5M Tris-HCl, pH 8.8 - 3.75 ml
10% SDS 150 μl 150 μl
diH20 9 ml 11.03-(0.5×X) ml
TEMED 15 μl 7.5 μl
10% APS 75 μl 75 μl
Total Volume 15 ml 15 ml