RNA purification

From Genome@Yale

Before starting, make sure pipetmans and bench are RNase free. Spray down pipetman with RNaseZAP and replace bench paper towel.

Trizol Information

For 10cm plates (75cm2 flasks)

1. Aspirate media
2. Add 1 mL Trizol and swirl around to detach cells
3. Transfer Trizol-cell suspension to 1.5mL Eppendorf tubes
4. Add 200uL chloroform
5. Vortex 15 seconds
6. 2 minutes at RT
7. Spin 14K rpm 15 minutes at 4C
8. Transfer aqueous (upper) phase to new sterile tubes containing 0.5mL isopropanol
9. 10 minutes at RT
10. Spin 14K rpm 15 minutes at 4C
11. Wash 1X with 75% EtOH
12. Spin 10K rpm 5 minutes at 4C
13. Air dry pellet
14. Resuspend in 50uL dH2O (DEPC treated or RNase free)
15. Add 1.25uL RNasin
16. OD RNA samples
17. Load 2ug sample to agarose gel to check RNA quality (or on Bioanalyzer)
18. Add 3X volume of 100% EtOH to RNA samples and store at -80C

Before use, RNA is precipitated by addition of 1/40 volume of sodium acetate and centrifugation at at 15000 rpm.

The RNA pellet is resuspended in desired volume of RNase free dH2O.

The RNA can be further processed using Qiagen RNeasy RNA cleanup kit to remove the trace amounts of Trizol, chloroform and other contaminants.