Procedure

1. Preheat Extraction Buffer at 60°C.
2. Weigh 100 mg of fresh leaf tissue and grind it to powder in Liquid Nitrogen in a chilled mortar and pestle.
3. Add 0.5 ml of TE buffer to the tube.
4. Centrifuge at 500 g for 10 min at room temperature.
5. Remove the supernatant and wash the pellet twice with 0.5 ml Wash buffer I.
6. Finally suspend the pellet in 0.5ml Wash buffer II.
7. Add 100 µg/ml Proteinase K to the above suspension.
8. Add 1.0 ml pre warmed Extraction buffer.
9. Incubate the sample at 60°C for 30 min with gentle shaking.
10. Allow the sample to cool for 2 - 3 mins.
11. Add 1.0 ml of Chloroform: Isoamyl Alcohol (24: 1) to the tube.
12. Centrifuge at 12,000 X g at room temperature for 15 min.
13. Transfer the upper aqueous phase to a new tube and repeat the process twice or thrice.
14. Transfer the upper aqueous phase to a new tube and add two volume of cold 100% ethanol and 0.1 volume of 3 M Sodium acetate pH 5.2 to precipitate the DNA.
15. Incubate at -20°C for 4 – 5 hours or overnight for precipitation of DNA.
16. Centrifuge at 12,000 X g for 10 min at 4°C to pellet the DNA.
17. Wash the DNA pellet with 70% Ethanol.
18. Centrifuge at 8000 X g for 5 min at 4°C to pellet the DNA.
19. Repeat the washes twice or thrice.

20. Dry the pellet and resuspend the DNA pellet in 100 μl of TE.

Solutions
Wash Buffer I

50 mM Tris HCl pH 8.0
5 mM EDTA pH 8.0
50 mM NaCl

Wash Buffer II

50 mM Tris HCl pH 8.0
50 mM EDTA pH 8.0

Extraction Buffer

3% CTAB
1% (w/v) Sarkosyl
20 mM EDTA
1.4 M NaCl
0.1 M Tris HCl
1% (v/v) β-Mercaptoethanol

TE Buffer

10 mM Tris HCl pH 8.0
1 mM EDTA pH 8.0