1. Grind leaves (0.5 to 5 g, fresh or liquid N₂-frozen) in liquid nitrogen with mortar and pestle into a fine powder.

2. Transfer the powder to a centrifuge tube containing 1 to 10 ml of extraction solution. Mix quickly.

3. Extract with phenol/chloroform/isoamyl alcohol (PCI) for three times. (Spin at 1000 to 5000 X g for 20 to 30 min)

4. Precipitate with 2 vol of ethanol and 0.1 vol of 3 M Na-acetate (pH 5.2) at -20^oC overnight.

5. Spin at 3000 X g for 20 min. Dissolve pellet in 1 to 4 ml of 4 M guanidine solution.

6. Precipitate with 0.5 vol of ethanol by adding ethanol drop-wise while vortexing the tube slowly. Keep on ice for 1 hr.

7. Spin at 5000 X g for 20 min. Discard supernatant. Dry pellet briefly. Dissolve pellet in 1 to 4 ml of DEPC-water. (If it is hard to dissolve, put the tube in 68^oC water bath)

8. Extract with PCI once, chloroform/isoamyl alcohol once.

9. Precipitate with 2.5 vol of ethanol and 0.1 vol of 3 M Na-acetate (pH 5.2) at -20^oC overnight.

10 . Dissolve in 100 to 500 ul of DEPC-water.

Solutions

1. Extraction Solution (500 ml)

Tris-HCl, pH 9.0                                 0.1 M (50 ml 1 M)
NaCl                                                   0.1 M (10 ml 5 M)
EDTA                                                 5 mM (5 ml 0.5 M)
SDS                                                    1 % (50 ml 10%)
VRC (Add freshly) 10 mM

guanidine-HCl                                     4 M (38.21 g)
EDTA                                                  10 mM (2 ml 0.5 M)
Tris-HCl, pH 7.0                                  0.1 M (10 ml 1 M)