Protocol 2: Total RNA preparation

 

  1. Take cells out of -80°C freezer and add 3 ml of acid phenol-chloroform-isoamylalcohol (125:24:1, pH4.7; Sigma P-1944), pre-warmed for 10 minutes at 65°C and 3 ml of TES (10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS; autoclaved) per 200 OD600 units to the RNA. For 50 OD600 units add 0.7 ml of Phenol-Chloroform + 0.7 ml of TES.

  2. Vortex tube at highest setting at ~ 20° angle, to resuspend pellet.

  3. Incubate at 65°C in waterbath for 1 hr. Incubate in beaker filled with hot water (move beaker to vortexer, etc.). Vortex 20 seconds (10 seconds upright, 10 seconds at 20° angle) every 10 minutes.

  4. Vortex 20 seconds, then aliquot into 4x 1.5 ml Eppendorf tubes. Spin 20 minutes at 14,000 rpm at 4°C.

  5. Extract with 750 µl of acid phenol-chloroform-isoamylalcohol per tube. Vortex 20 seconds (10 seconds up, 10 seconds angle). Spin 10 minutes at 4°C.

  6. Extract with 24:1 chloroform:Isoamyl alcohol (Sigma C-0549). Vortex 20 seconds, spin ~ 10 minutes room temperature.

  7. Transfer aqueous phase to tube with 50 µl of 3M Sodium Acetate (NaOAc) pH 5.2. Add 1ml of 100% EtOH (pre-cooled to -20°C), fill to top of tube. Store at -20°C for longer than 1/2 hr.

  8. Spin down RNA for 5 minutes in a microcentrifuge at room temperature. Aspirate (leave a little left).

  9. Wash with 500 µl of 80% ETOH (temperature ~ -20°C), no shaking, just add. Spin down for 1 minute.

  10. Aspirate, remove last bit with a pipette. Let air dry 1 minute.

  11. Add DEPC treated water to samples to resuspend. To calculate amount to add: expect 20 µg RNA for a heat-shocked culture or 30µg RNA for a 30°C grown culture per unit OD600.

  12. Measure OD260 / OD280 accurately (e.g. by measuring 5 µl of a 1:20 dilution).

  13. Make aliquots, snap-freeze and store at -80°C.

Resuspension of samples: