Cetyltrimethylammoniumbromide(CTAB)Pla

Procedure

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℃ 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).

22. Discard the supernatant.

23. Centrifuge for 5 additional min and remove as much of the remaining liquid as possible with a pipette. Do not dry the pellet.

23. Add 100 to 250 μl TE Buffer. Dissolve the pellet by gentle pipetting.

Solutions Phenol:Chloroform 1:1 Phenol:Chloroform 4 M Lithium Acetate 3 M Sodium Acetate TE Buffer 1 mM EDTA, pH 8.0 10 mM Tris CTAB Buffer 800 mM NaCl 1% (v/v) Sarkosyl 140 mM Sorbitol Autoclave 22 mM EDTA 220 mM Tris, pH 8.0 0.8% (v/v) CTAB

BioReagents and Chemicals Cetyltrimethylammonium Bromide Sarkosyl Chloroform Phenol Sodium Acetate Ethanol Sodium Chloride EDTA Sorbitol Isopropanol Tris Lithium Acetate