Cetyltrimethylammonium bromide (CTAB) Plant DNA Extraction

Friday, November 21, 2003

Description
Protocol for Cetyltrimethylammonium bromide (CTAB) Plant DNA Extraction

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 65C 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 l of TE Buffer by gentle pipetting.

16. Add 100 l 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 l TE Buffer. Dissolve the pellet by gentle pipetting.

Recipes
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
220 mM Tris, pH 8.0
22 mM EDTA
0.8% (v/v) CTAB

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Cetyltrimethylammonium bromide (CTAB) Plant DNA Extraction