RNA EXTRACTION FROM ARABIDOPSIS

using TRIZOL; for small-scale preps.

(Mike Deyholos, 13 Jan 00; based on LifeTechnologies protocols)

See product insert for details; (click on 'Protocols' at http://search.lifetech.com/).

1. Homogenize 100mg-200mg of Arabidopsis tissue in liquid nitrogen.

2. Combine homogenized tissue with 1mL of TRIzol reagent in an RNase-free microfuge tube.

3. Grind briefly with a disposable blue pestle (e.g. Kimble)

4. Incubate 5 min at room temp.

5. Add 0.2mL chloroform.

6. Shake by hand for 15 sec.

7. Incubate 3 min at room temp.

8. Centrifuge at 12 000 x g for 10 min at 2 to 8'C

9. Transfer the (upper, colourless) aqueous phase to a fresh RNase free tube.

10. Precipitate by adding 0.5mL isopropryl alcohol to aqueous phase.

11. Incubate at room temp for 10 min.

12. Centrifuge at 12 000 x g for 10 min.

13. A very small pellet should be visible at the bottom of the tube. Decant the isoproyl alcohol, and wash the pellet with 1mL of 75% ethanol (use RNase-free water to make the 75% ethanol).

14. Centrifuge at 7 500 x g for 10 min.

15. Decant the alcohol, and invert the tube on a Kimwipe to draw off residual liquid.

16. Allow the pellet to air-dry for 5-10 min (e.g. until all of the droplet of EtOh have disappeared from the walls of the tube), but DO NOT ALLOW THE PELLET TO FULLY DRY OUT.

17. Resuspend pellet in ~10uL RNase-free water. Freeze at -20'C for short-term storage (e.g. <1 month), or -80'C for longer term storage.

18. Yield may be quantified fluorometrically, e.g. using RiboGreen (Molecular Probes).