Table 7.
Recipe to prepare solution for extraction of mitoribosomes. This solution can be made at the same time as preparing the sucrose gradient buffers. Ensure the digitonin used is high-purity and is prepared the same day as the experiment.
| Extraction Buffer | |||
|---|---|---|---|
| Reagent | [Stock] | [Final] |
1x Volume
(500 μL) |
| Tris pH 7.4 | 1 M | 10 mM | 5 μL |
| NH4Cl | 1 M | 100 mM | 50 μL |
| MgCl2 | 1 M | 10 mM | 5 μL |
| PMSF | 100 mM | 1 mM | 5 μL |
| DTT | 100 mM | 0.25 mM | 1.25 μL |
| Digitonin (high purity) | 10 % | 0.6 % | 30 μL |
| Yeast protease inhibitor cocktail | 100x | 1x | 5 μL |
| RNase inhibitor | 40 U/μL | 0.1 U/μL | 1.25 μL |
| H2O | - | - | 397.5 μL |