In vitro translation --- Rabbit Reticulocyte Lysate

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.