Protocol for making Gels (recipe for 4 or 2 gels)
1. Clean the plates with glass cleaner and then ethanol. Then place the plates in the casting
stands.
2. Prepare the 12% resolving gel. Combine everything except the APS and TEMED.
4 gels
2 gels
H2O
8.8 mL
4.4 mL
40% acrylamide mix
6 mL
3 mL
1.5 M Tris
5 mL
2.5 mL
10% SDS
200 L
100 L
3. Pipette out 1.6 mL of this mix into a separate beaker. Then add 8 L of APS and 8 L of
TEMED. Next, pipette 200 L of this mixture into every plate. Make sure to pipette this
into the plates quickly after adding the TEMED because it will cause the mixture to
solidify if not done in a timely manner. This will serve as a seal to prevent leaks from the
resolving gel.
4. Add APS and TEMED to the original resolving gel mix.
4 gels
10% APS (ammonium
200 L
persulfate
TEMED
8 L
2 gels
100 L
4 L
5. Quickly add this mixture to the plates. Each plate should be at least half filled with the
resolving gel if not more. Pour ethanol in every plate after adding the resolving gel to
the plates. This will keep the resolving gel from solidifying with warped or curved edges.
6. Make the 5% stacking gel. Do not add the TEMED until you have poured the ethanol out
of every plate.
4 gels
2 gels
H2O
5.5 mL
2.75 mL
40% acrylamide mix
0.950 mL
0.475 mL
1.0 M Tris
0.950 mL
0.475 mL
10% SDS
75 L
37.5 L
10% APS
75 L
37.5 L
TEMED
8 L
4 L
7. Top off every plate with the resolving gel. Insert a comb into every plate quickly after. Once
the plates have solidified, lightly rinse them with water and wrap each in a damp paper
towel. Store in the refrigerator.