VPB 321: Animal Biotechnology (2+1)

ISOLATION OF RNA

Aim

• To isolate RNA from yeast.

Material required

• Phenol solution (900 g/l), Potassium acetate (200 g/l), Diethyl ether, Yeast.

Procedure

• 30 ug dried yeast suspension is made in 120 ul of water and kept at 37 ° C for 15 minutes. 160 ul of Phenol is added to this suspension. After stirring the suspension for 30 minutes at room temperature, the solution is centrifuged at 1500 rpm for 15 minutes at 4 ° C.
• The upper aqueous phase is again subjected to centrifugation to sediment the denatured protein. Potassium acetate is then added to the final concentration of 20 mg/ l and RNA is precipitated by adding 2 volumes of ethanol.
• The solution is kept in ice for 1 hour. The solution is centrifuged for 5 minutes at 4 ° C. Wash the RNA with ethanol-water (3:1), ethanol and finally with ether, air dry and dissolve in buffered saline.
• The purity of the RNA is checked by measuring OD at 260 and280 nm using UV-spectrophotometer
• Good quality RNA will have a ratio of 1.8 to 2.0 at 260/280 nm

Questions

• Write the principle of RNA extraction from yeast.
• How to determine the purity and quantity of RNA in the solution?