1. Total RNA Isolation from cultured cells.
1. Wash cultured cells 2X with 8 ml PBS (without calcium, without magnesium) at RT.(For 100 mm dish use 5 ml PBS)
2. Add 8 ml TRIzol reagent (Gibco/BRL) to cells in T-75 flask.
(For 100 mm dish use 5 ml TRIzol reagent.)
3. Transfer lysed cell solution to a 15 ml flacon tube.
4. Incubate at RT for 5 minutes.
5. Add 1.6 ml Chloroform:Iso-Amyl alcohol (24:1) to each tube.
6. Shake vigorously for 30 seconds.
7. Incubate at RT for 3 minutes.
8. Spin in centrifuge at 4oC for 15 minutes at 4000 RPM.
9. Transfer upper phase (clear solution only; no white goop) to a new 15 ml tube.
10. Add 4 ml of Isopropanol (2-propanol) to each tube.
11. Mix by inverting several times.
12. Incubate at RT for 10 minutes.
13. Spin for 30 minutes at 4oC at 4000 RPM.
14. Remove supernatant with a pipette very carefully.
15. Resuspend pelleted RNA using pipette in 1 ml of 70 % Ethanol.
16. Transfer to a microfuge tube.
17. Spin for 10 minutes at 4oC at 14,000 RPM.
18. Remove supernatant with a pipette.
19. Aspirate remaining drops.
20. Air-dry pellet for 5-10 minutes at RT.
21. Resuspend RNA pellet in 80 ul DEPC treated water.
22. Spec 2 ul in 198 ul of water.
23. Store RNA at -80oC.
Jeffrey D. Randall, Takumi Yoshida, Ryoji Kojima, Steven Gullans © Page 2 9/6/2001
Jeffrey D. Randall, Takumi Yoshida, Ryoji Kojima, Steven Gullans © Page 2 9/6/2001