RNA purification using TRizol TRIZOL法提取RNA【University of Florida】

2. Phase Separation

a. Incubate samples (from 1g) for 5 minutes at room temperature.

b. Add 0.2 ml of chloroform to each tube.

b. Cap each tube. Shake samples vigorously by hand for 15 seconds.

c. Incubate samples for 5 minutes at room temperature.

d. Centrifuge samples for 15 minutes at 12,000 x g at 4^oC.

3. RNA Precipitation

a. Transfer the upper aqueous phase to a fresh tube.

b. Add 0.5 ml of isopropyl alcohol to precipitate RNA. If this RNA will be used for RT-PCR, first add 50 μl isopropyl alcohol, mix, incubate the samples at room temperature for 5 minutes and centrifuge at 12,000 x g for 10 minutes at 4^oC. Transfer the sample in a new tube.

c. Incubate for 5-10 minutes at room temperature.

d. Centrifuge for 10 minutes at 12,000 x g at 4^oC. The RNA will form a pellet on the side or bottom of the tube.

4. RNA Wash

a. Discard the supernatant.

b. Wash pellet with 1 ml 75% ethanol.

c. Mix sample by vortexing. The RNA pellet may float.

d. Centrifuge at 12000 x g for 5 minutes at 4^oC. If this RNA will be used for RT-PCR repeat steps a,b and c twice.

e. RNA pellet may be stored in ethanol at -70^oC for months.

5. Redissolving the RNA

a. Remove supernatant.

b. Air dry the pellet for 5-10 minutes. Do not completely dry out the pellet.

c. Dissolve pellet in 30 to 60 μl RNase free water or 0.5% SDS by passing the solution through a pipette tip and incubating for 10 minutes at 55-60^oC.

6. Determination of