| Tris-HCl (pH 7.5) |
20 mm |
| KCl |
50 mm |
| MgCl2 |
10 mm |
| Cycloheximide |
100 µg/mL |
| Dithiothreitol (DTT) |
1 mm |
| cOmplete protease inhibitor cocktail tablets, EDTA-free (Roche) |
2 tablets/100 mL culture |
| Heparin |
0.2 mg/mL |
| RNasin (Promega) |
1 µL/mL |
| Phenylmethylsulfonyl fluoride (PMSF) |
1 mm |
| Combine the first three ingredients. (If desired, a 10× stock solution containing 200 mm Tris-HCl, 500 mm KCl, and 100 mm MgCl2 can be prepared and stored on a long-term basis.) Just before use, add the cycloheximide and DTT, as well as the protease
inhibitor tablets, heparin, and RNasin (the latter three are optional but highly recommended). For processing meiotic cells,
include PMSF.
|
