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|  | | | 1. Grind 2 to 5 g of frozen leaves to a very fine powder with a liquid nitrogen-cooled mortar and pestle.
2. Add 25 ml of CTAB Buffer and transfer to a 50 ml tube.
3. Incubate at 65°C for 20 min with occasional vigorous shaking.
4. Add 10 ml of Chloroform, shake well, and place on a tube inverter at room temperature for 20 min.
5. Centrifuge at 1,000 X g for 5 min to resolve phases.
6. Transfer the aqueous phase to a fresh tube, add 17 ml of Isopropanol, mix, and place on ice for 10 min.
7. Centrifuge at 1,000 X g for 5 min to collect the precipitate.
8. Discard the supernatant and dry the inside of the tube with a paper towel (do not dry the pellet).
9. Add 4 ml of TE Buffer and dissolve the precipitate by gentle inversion.
10. Add 4 ml of 4 M Lithium Acetate and incubate on ice for 20 min.
11. Centrifuge at 1,000 X g for 10 min.
12. Transfer the supernatant to a fresh tube, add 16 ml of 100% Ethanol, and incubate on ice for 20 min.
13. Centrifuge at 1,000 X g for 5 min to collect the precipitate.
14. Discard the supernatant and dry the inside of the tube with a paper towel (do not dry the pellet).
15. Dissolve DNA in 900 μl of TE Buffer by gentle pipetting.
16. Add 100 μl of 3 M Sodium Acetate. Divide the sample evenly into two microcentrifuge tubes.
17. Add an equal volume of Phenol to each tube, mix well, centrifuge to separate the phases (1,000 X g), and save the aqueous phase (upper phase).
18. Add an equal volume of Phenol:Chloroform to each tube, mix well, centrifuge to separate the phases (1,000 X g), and save the aqueous phase (upper phase).
19. Add an equal volume of Chloroform to each tube, mix well, centrifuge to separate the phases (1,000 X g), and save the aqueous phase (upper phase).
20. Add 2 volumes of 100% Ethanol to each tube and incubate on ice for 5 min.
21. Centrifuge 5 min to collect the precipitate (approximately 5,000 X g).
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