RNA EXTRACTION FROM ARABIDOPSIS AND ICE PLANT

for large preps

(M. Deyholos, 21 March, 2000)

Based on Krapp et. al. (1993) Plant Journal 3:817.

1. Grind tissue in liquid nitrogen.

2. Add 1-2 volumes of extraction buffer.

3. To the total volume of extraction buffer and tissue, add 1 volume of phenol:chloroform:isoamyl (25:24:1) and shake briefly.

4. Spin 15min at 8000xg (e.g. 8500 rpm in 30ml COREX tubes in a Sorvall SS-34 rotor).

5. If the upper phase is cloudy, repeat the phenol:chloroform:isoamyl extraction.

6. Transfer the upper, aqueous phase to a fresh tube, and add 1/10 volume 3M sodium acetate (RNase free), and 2 volumes of ethanol.

7. Spin 15min at 8000xg.

8. Discard the supernatant. Wash the white pellet with 70% ethanol (made with RNase-free water).

9. Drain the ethanol from the tube and resuspend the pellet in 1-2ml of resuspension buffer.

10. Incubate at 4’C for at least 1 hour.

11. Spin at 10 000xg (e.g. in 1.5mL eppendorf tubes) for 15 min.

12. Rinse the pellet in 70% ethanol, air-dry briefly, and resuspend in a small volume of RNase-free water.

extraction buffer

for 200mL

8M Guanidine-HCl 152.8g

20mM MES 0.78g

20mM EDTA 8mL (0.5M stock)

50mM b-ME 694mL bottle strength

sterile water to 200mL; pH to 7.0

resuspension buffer

for 50mL

2M LiCl 1.0g

10mM sodium acetate 167mL (3.0M stock)

RNase-free water to 50mL; pH to 5.2