Preparing a SDS-PAGE Gel
Contents
SOP
Separating Gel
- For 12 mL final volume (2 gels)
- Combine, adding APS and TEMED last
- Pour into cassette, overlay with the top layer of water saturated butanol
| Component | 6% | 7.5% | 10% | 12.5% |
|---|---|---|---|---|
| SDS-PAGE Separating Gel Buffer | 3 mL | 3 mL | 3 mL | 3 mL |
| Acrylamide Solution | 2.4 mL | 3 mL | 4 mL | 5 mL |
| Water | 6.6 mL | 6 mL | 5 mL | 4mL |
| APS (make fresh, 0.1g/mL in water) | 200uL | 200 uL | 200 uL | 200 uL |
| TEMED | 30 uL | 30 uL | 30 uL | 30uL |
- For a Phos-Tag Gel, add 24 uL 5 mM Phos-Tag and 24 uL 10 mM MnCl in the separating gel only
Stacking Gel
- Rinse polymerized separating gel with water
- For 10 mL (2 gels)
- Combine, adding APS and TEMED last
- Pour over separating gel and insert comb
| Component | |
|---|---|
| SDS-PAGE Stacking Gel Buffer | 2.5 mL |
| Acrylamide Solution | 1.7 mL |
| Water | 4 mL |
| APS (make fresh, 0.1g/mL in water) | 200 uL |
| TEMED | 30 uL |