(for culture cells, in 100 mm dish)
1.Wash with nuclease free PBS once.
2. Add 1 ml trizol, and then scrub the dish bottom.
3. Transferred the lysate to 1.5 ml eppendorf tube, and keep on ice.
4. Add 0.2 ml chloroform, and vertex for 30 seconds.
5. Incubate on ice for 5-10 minutes.
6. Centrifuge at 14000 rpm for 15 minutes at 4 centigrade.
7. Transfer aqueous phase to new eppendorf
8. Add 0.5 ml isopropanol.
9. Incubate on ice for 10 minutes.
10.Centrifuge at 14000 rpm for 15 minutes at 4 centigrade.
11.Discard the supernatant. The pricipitated pellet is raw RNA.
12.Wash the RNA pellet with 0.5 ml 75% ethanol. Centrifuge at
9000 rpm for 5 minutes at 4 centigrade. Discard the supernatant.
13.Repeat step 13 one more.
14.Air dry the pellet for about 10 minutes. (Note: don't overdry,
otherwise, it will be very difficult to dissolve it.)
15.Rehydrate with 20-50 micro liter DEPC water.