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This protocol uses Rabbit Reticulocyte Lysate from Promega.
1. Rapidly thaw the Rabbit Reticulocyte Lysate by hand-warming and place on ice.
Thaw other components at room temperature or 37 °C and place on ice.
2.
Make the following reaction mixture:
| Rabbit Reticulocyte Lysate |
33 ml |
| Amino Acid Mixture, Minus Methionine, 1 mM |
33 ml |
| 35S-methionine (10 mCi/ml) or 35S-EXPRESS |
2 ml |
| 25 mM Magnesium Acetate |
0-4 ml |
| 2.5 M Potasium Chloride (KCl) |
1.4 ml |
| 100 mM DTT |
0-1 ml |
| RNasin Ribonuclease inhibitor |
1 ml |
| RNA substrate |
1-12 ml |
| Nuclease-free ddH2O |
to 50 ml |
5. Mix and centrifuge to collect the reaction mixture on the bottom of the tube.
6. Incubate at 30 °C for 60-90 minutes.
7. Stop the reaction by add SDS-PAGE loading buffer or store at -20 °C.
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