3 g nodules (fresh or frozen in liquid N2) were ground to a powder in mortar and pestle with liquid N2. To the powder was added 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 Miracloth and rinse the cloth with 5 ml of the same buffer solution. Spin filtrate in a sterile centrifuge tube and spin 5 min 5 min at 1500 rpm. Respin supernatant 5 min at 9000 rpm. Resuspend pellet in LiCl buffer containing 10 mM vanadyl ribonucleosides and treat as stated above.

0.1 M LiCl, 1% SDS, 7 M urea, 0.1 M Tris (pH 8.4), 5 mM EDTA

Extract with phenol:chloroform 3 times. Precipitate with 2 vol ethanol. Spin. Redissolve ppt. in 2 ml H2O 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 ppt 3 times with ice cold 3 M Na acetate (6) treated with DEPC. Redissolve RNA in DEPC-treated H2O, ppt. with 2.5 vol ethanol, and store as ppt. at -80℃.

ISOLATION OF RNA FROM FREE-LIVING RHIZOBIA

Cells from 100 ml culture in early log phase GYPC were spun and resuspended in 1 ml sterile 25% sucrose. This solution was transferred to a sterile centrifuge tube and 6.5 ml M-STET, 0.5 ml lysozyme (10 mg/ml), and 0.4 ml 200 mM vanadyl ribonucleodides were added.

M-STET: 4% sucrose, 6% triton X100, 0.06 M Tris (pH 8), 0.06 M EDTA

Incubate on ice 5 min. Boil 2 min [or add SDS/sarkosyl, extract with phenol:chloroform, and ppt.], chill, and spin 10 min at 8000 rpm. To the supernatant add 0.7 vol isopropanol, chill, spin again, and resuspend pellet in 5 ml.

5X Running buffer for RNA (formaldehyde) gels: (For 500 ml) 2 ml 0.5 M EDTA, 6.8 g Na acetate, 12.8 g MOPS (free acid), 9.0 g MOPS (base).

Induce cells normally - 10 ml of 0.2 OD600.

Wash cells (grown O/N in ORS minimal plus yeast at 30 or 37℃) 2 times with nif media and diluting to 0.2 OD (10 mls each). Check 1 bottle for induction by acetylene reduction.

After induction add 0.5 ml vanadyl ribonucleosides directly through septum into vial with a syringe (vanadyl protects cells against oxygen).

Put cells in plastic tubes, spin a for 2 min at 8000 rpm in Sorvall.

Resuspend in 10 M urea lysis buffer with plastic pasteur pipette.

Add 0.5-0.75 ml of phenol:chloroform (50:50) ["phenol" is phenol:m-cresol:hydroxyquinoline] to microfuge tube.

Vortex, place at 65℃ for 2-3 min. repeat. vortex and spin 2-3 min in microfuge.

Re-extract liquid phase 2 more times at room temp with phenol:chloroform. Avoid proteinaceous interphase when removing upper liquid phase.

Extract with chloroform 1 time

Precipitate with 100% ethanol, 0.3 M Na acetate (5.2) at -20 C O/N.

Collect ppt., wash 1 time with 70% ethanol, 1 time with 100% ethanol, and resuspend in 10 mM Na acetate (5.2) - treat for gels.

formaldehyde gels:

see manual

run in cold room O/N at 15 V in minigel; unit

blot directly to nylon-66 membranes

deionizing formamide - use columns, do not add resin directly to formamide.

BLOTTING FORMALDEHYDE GELS

Blot 15-45 min with 50 mM NaOH (autoclaved).

Blot 3-5 hrs with 45 mM Na acetate (5.2).

Expose to uv lamp 2 min.

prehybridize with 10 mg/ml heparin 5 min - 4 hr.

hybridize O/N at 42℃.

Wash: 500 ml 5X SSPE 30-45 min at 42℃(46-54 setting) 500 ml 1X SSPE 0.1% SDS at 42℃ for 45-60 min. 500 ml 0.1 X SSPE 0.1% SDS room temp.

Rewash 1X SSPE

Blot dry (not totally dry! - do not air dry) put in saran wrap.

Autoradiograph. To strip:

10.Wash at 65℃ 1-2 hrs in 1/10 lysis buffer.