- Grind 3 g nodules (fresh or frozen in liquid N2) to a powder in mortar and
pestle with liquid N2.
- To the powder, add ice-cold 0.5 M mannitol (or sucrose), 0.05 M Tris
(pH 7.5), 0.02 M Na succinate, 5 mM Na dithionite.
- Pass the resuspension through one layer of cheesecloth and rinse the cloth
with 5 mL of the same buffer solution.
- Spin filtrate in a sterile centrifuge tube and spin 5 min at 1500 rpm.
- Respin supernatant for 5 min at 9000 rpm.
- Resuspend pellet in LiCl buffer containing 10 mM vanadyl ribonucleosides
and treat as stated above.
Extract with phenol
- 0.1 M LiCl
- 1% SDS
- 7 M urea
- 0.1 M Tris (pH 8.4)
- 5 mM EDTA
: chloroform 3 times. Precipitate with 2 vol ethanol.
Spin. Redissolve precipitate in 2 mL H2
O treated with 0.1% DEPC and
autoclaved. Add 6 mL 4 M Na acetate (6) treated with DEPC. Incubate on ice
2 hr, spin 30 min at 8000 rpm, and wash precipitate 3 times with ice cold 3
M Na acetate (6) treated with DEPC. Redissolve RNA in DEPC-treated H2
precipitate with 2.5 vol ethanol, and store as precipitate at –80°C.