Isolation of RNA

Isolation of RNA

Aim

To isolate RNA from yeast

Principle

Total yeast RNA is obtained by extracting with phenol.  The concentrated phenol causes denaturation of protein.  The turbid suspension was centrifuged and the lower phenol phase contain DNA and the upper aqueous phase contains carbohydrate and RNA.  RNA is then precipitated with alcohol.  The product obtained will be free of DNA but usually contaminated with polysaccharides.  Further purification may be done by treating with amylase.

Materials required

1. Yeast

2. Phenol

3. Sodium acetate

4. Absolute alcohol

5. Diethyl ether

6. Water bath at 37^oC

Procedure

1. 3 gm of yeast was suspended in 12 ml of water previously heated at 37^oC.

2. It was left for 15 minutes at this temperature and 16 ml of concentrated phenol was added

3. The suspension was mixed mechanically for 30 minutes at room temperature.  It was centrifuged at 3000 rpm for 15 minutes in cold.

4. The aqueous upper layer was removed carefully with a Pasteur pipette and centrifuged at 10000 rpm for 15 minutes in a cooling centrifuge to sediment denatured protein

5. Sodium acetate was added to the supernatant at a final concentration of 20 gm/L and the RNA was precipitated by adding two volumes of ethanol.  The solution was cooled in ice and left to stand for 1 hour.

6. The precipitate was collected by centrifuging at 2000 rpm for 5 minutes in cold.  

7. The RNA was washed with ethanol:water (3:1), ethanol and finally ether, and air dried.

Result

RNA from yeast was isolated