RNA preparation

Day 1

1. Grind 6g of Arabidopsis leaves in liquid N2 with mortar and pestle.

2. Transfer to 50ml Falcon tube.

3. Add 18ml of 1:2:1 solution (precipitation at RT)

1 volume of 2M Tris-HCl pH 8.2

2 volumes of 0.5M EDTA pH 8.0 (DEPC treated)

1 volume of 20% SDS

4. Add 9ml of phenol saturated with 0.1M Tris-HCl pH 8.2. Stir for minimum 15'.

5. Add 9ml chloroform. Stir for minimum 10'.

6. Centrifuge 4500rpm 15'.

7. Treat upper phase (aqueous) twice with 9ml chloroform, centrifuging 4500rpm 15'.

8. Transfer 15 ml of aqueous phase to 30ml disposable polypropylene tube and add equal volume of ice-cold 6M LiCl. Precipitate O/N on ice at 4°C.

Day 2

9. Centrifuge 9000rpm 20' 4°C.

10. Resuspend into 2.5ml H2O, add 0.25ml 3M NaAcetate pH 5.2 and 3 volumes of EtOH. Precipitate at RT for ca 60min.

11. Centrifuge 9000rpm 20' 16°C, rinse with 70% EtOH, centrifuge 9000rpm 5', air dry pellet and resuspend in 500µl H2O. Take 5µl for OD260/280 readings.

12. Store RNA at -20°C.