Friday, November 21, 2003

Description
TMV is a helical rod with two components, an RNA of 6.1 kb and a protein of 17.5 kDa. Ninety-five percent of the mass is protein and 5% of the mass is RNA. To isolate the RNA you dissociate the two components with hot detergent (SDS) and partition the protein into Phenol. You harvest the RNA from the aqueous phase by adding Ethanol.

Procedure
1. Dilute the TMV 10-fold in 2X NET or 2X LET (see #2).

2. Add 0.1 volume of 10% SDS.

3. Heat in a clear beaker of hot ddH2O (70C) until the cloudiness from light scattering disappears indicating disruption of the virus structure. Cool the solution to room temperature.

4. Add an equal volume of Water-Saturated Phenol (CAUTION! see #1) and mix by vigorously vortexing.

5. Centrifuge to separate the aqueous and Phenol phases.

6. Recover the top aqueous layer and add an equal volume of Water-Saturated Phenol and mix by vigorously vortexing. Centrifuge to separate the aqueous and Phenol phases and recover the top aqueous layer.

7. Add 2.5 volumes of Ethanol to the aqueous phase in a baked siliconized corex tube for volumes over 1.5 ml or a microcentrifuge tube for smaller volumes. Centrifuge for 30 min at 10,000 rpm in a bench-top centrifuge (or at full speed in a microcentrifuge).

8. Remove the supernatant and add an equal amount of 96% Ethanol to the tube (see #3).

9. Remove the supernatant and allow the Ethanol to evaporate. Dissolve the pellet in a minimal volume of ddH2O (see #4)

10. Read the absorbance at 260 nm. A 1 mg/ml concentration of RNA reads 23 at 260 nm (see #5).


Recipes
LET 20 mM Tris
0.2 M LiCl
2 mM EDTA


NET 0.2 M NaCl
20 mM Tris
2 mM EDTA


Water-Saturated Phenol (CAUTION! see Hint #1) See protocol for the Preparation of Water-Saturated Phenol


96% (v/v) Ethanol

10% (w/v) SDS


Supplies


Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The TMV is usually supplied as a suspension at 50 to 100 mg/ml in Phosphate Buffer and needs to be diluted or dialyzed in order to proceed.

3. Centrifuge briefly if the pellet becomes dislodged or breaks apart.

4. The desired final RNA concentration is 2 to 10 mg/ml.

5. Be careful, many compounds (particularly Phenol) absorb 260 nm UV light which can lead to erroneous RNA concentration values.