In vitro transcription/translation --- TNT

This protocol uses the TNT coupled transcription and translation Kit.

1. Rapidly thaw the TNT Quick Master Mix by hand-warming and place on ice. Thaw other components at room temperature and place on ice.

2. Plasmid must have been linearized. Both plasmid and PCR product must have the appropriate promoter site (T3, T7, or Sp6).

3. Use a TNT kit appropriate for the promoter. Make the following reaction mixture:

TNT Quick Master Mix 40 ml
35S-methionine (10 mCi/ml) or 35S-EXPRESS 2 ml
Plasmid or PCR DNA (0.5-1 mg) 2 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.