Phenol/Freeze RNA Prep (Yeast)

Tuesday, November 18, 2003

Description
Phenol/Freeze RNA Prep (Yeast)

Procedure
1. Grow 10 ml of yeast to 5E6 cells/ml.

2. Harvest cells by centrifugation and resuspend cells in 400 l of AE buffer.

3. Transfer cells to a 1.5 ml microcentrifuge tube and add 40 l of 10% SDS. Vortex.

4. Add equal volume of phenol, vortex, and incubate at 65 deg C for 4 minutes.

5. Rapidly chill tube in dry ice/ethanol bath until phenol crystals appear, and centrifuge at room temperature for 2 minutes.

6. Transfer aqueous phase to a new tube, add an equal volume of phenol/chloroform/IAA, vortex, and spin for 5 minutes at room temperature.

7. Transfer aqueous phase to a new tube, add 1/10 volume of 3M Na Acetate, pH 5.3, 2.5 volumes of ethanol, and then precipitate for 20 minutes at -20 deg C.

8. Pellet RNA, wash with 80% ethanol, air dry pellet, and resuspend in 20 l water.

Recipes
(DEPC treatment means: add DEPC to 0.1%, mix O/N, autoclave to destroy DEPC)

DEPC treated water

AE buffer (DEPC treat)
50mM Na acetate, pH 5.3
10mM EDTA

10% SDS

Phenol, equilibrated with AE buffer

Phenol:chloroform:isoamyl alcohol, 25:24:1

3M Na acetate pH 5.3 (DEPC treat)

RNase-free 95% EtOH (-20 deg C)

RNase-free 80% EtOH

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Phenol/Freeze RNA Prep (Yeast)