Brief Description
Seperating gel 5% 7% 9% 12.5% 15%
Protocol Details
Lower Gel
| Reagents | 5% | 7% | 9% | 12.50% | 15% |
| 2 minigels | 2 minigels | 2 minigels | 2 minigels | 2 minigels | |
| 30% Acrylamide mix | 1.7 ml | 2.4 ml | 3.0 ml | 4.2 ml | 5 ml |
| 4xLGB | 2.5 ml | 2.5 ml | 2.5 ml | 2.5 ml | 2.5 ml |
| 20% SDS | 50 ul | 50 ul | 50 ul | 50 ul | 50 ul |
| Degas Water | 5.7 ml | 5.0 ml | 4.4 ml | 3.2 ml | 3.2 ml |
| 10% APS (0.1g/ml; fresh) | 50 ul | 50 ul | 50 ul | 50 ul | 50 ul |
| TEMED (undil) | 3.3 ul | 3.3 ul | 3.3 ul | 3.3 ul | 3.3 ul |
Stacking Gel Buffer (SGB, 0.5 M Tris)
12.1 g Tris up to 200 ml of H2O, PH 6.8 (store in 4C)
Lower Gel Buffer (LGB, 1.5 M Tris)
36.3 g Tris, up to 200 ml of H2O, PH 8.8 (store in 4C)
20% Sodium Dodecyl Sulfate (SDS)
20 g SDS, up to 100 ml in H2O
1.5% Ammonium Persulfate (APS)
1.5 g APS, up to 100 ml in H2O (Aliquot and store in -20C)
Prepare Plates:
Ensure that everything is clean (Best way to ensure no leakage!)
Pour lower gel:
- Combine Acrylamide, water, SDS, LGB
- Mix by swirling (don't create bubbles)
- Add APS and TEMED (swirl to mix)
- Pipet mixture between plates to about 2/3 up with enough room for a few cm's from the bottom of the comb (move quickly)
- Add water or 70% EtOH to make top smooth.
- wait about 20 min.
- Pipet Upper Gel displacing water/EtOH to about 1-2cm from top.
- Insert comb displacing the rest of the water/EtOH.
- Let sit for about 15 min.