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Star Republic: Guide for Biologists

Yeast RNA prep --- phenol

1. Acidic and hot phenol is used for breaking cells and purifying RNA.

2. Harvest 10 ml mid-log phase cells.

3. Resuspend cells in 0.4 ml of 50 mM sodium acetate (pH5.3), 10 mM EDTA.

4. Immediately freeze in liquid nitrogen.

5. Add equal volume of phenol. Alternatively, resuspend cells in 10 mM Tris-Cl pH 7.4, 10 mM EDTA, 0.5% SDS, freeze, and add acid phenol (water-saturated, not TE-saturated).

6. Vortex.

7. Incubate at 65 C for 10-60 minutes.

8. Cool on ice for 5 minutes.

9. Centrifuge at maximum speed in a microfuge for 5 minutes at 4 C.

10. Transfer aqueous phase to a new tube.

11. (optional) Repeat extraction with acid phenol without heating and cooling until the interface is clean.

12. Extract once with phenol:chloroform: isoamyl alcohol (25:24:1).

13. Add 1/10 volume of 3M sodium acetate (pH 5.3) and 2 volumes of ethanol.

14. Precipitate at -20 C for > 20 minutes.

15. Pellet RNA, wash with 70% ethanol, and air-dry.

16. Resuspend RNA in 20 ml of nuclease-free ddH2O or TE.