Overview

Procedure

1. Preheat the CTAB Isolation Buffer at 60℃.

2. Grind 2 g of fresh, leaf tissue to a powder in Liquid Nitrogen in a chilled mortar and pestle.

3. Scrape the powder into a chilled 50 ml tube.

4. Add 3 to 5 ml CTAB Buffer per gram tissue to the tube.

5. Incubate the sample at 60℃ for 30 min with occasional gentle swirling.

6. Add the same volume of Phenol:Chloroform to the sample, gently swirling.

7. Centrifuge at 16,000 X g at room temperature for 10 min.

8. Remove the yellow, aqueous phase with a wide-bore pipette to a new 50 ml tube (see Hint #1).

9. Add the same volume of Chloroform:Isoamyl Alcohol to the tube, gently swirling.

10. Centrifuge at 16,000 X g at room temperature for 10 min.

11. Transfer the upper, aqueous phase to a new 50 ml tube.

12. Add two-thirds volume of cold 100% Isopropanol to the tube and mix gently to precipitate the nucleic acids (see Hint #2).

13. Centrifuge at 5,000 X g at room temperature for 10 min.

14. If any strands of DNA are visible, spool the non pelleted DNA with a glass hook. Discard the supernatant.

15. Add 5 to 10 ml Wash Buffer to the DNA pellet, including the spooled DNA collected with the glass hook.

16. Incubate for a minimum of 20 min (see Hint #3).

17. Centrifuge at 16,000 X g for 10 min.

18. Pour off the supernatant and allow the DNA pellet to dry.

19. Resuspend the pellet in 2 to 3 ml TE (see Hint #4).

20. Add RNase A to a final concentration of 10 μg/ml.

21. Incubate the samples for 30 min at 37℃.

22. Extract once with an equal volume of Phenol:Chloroform. Mix well.

23. Centrifuge at 16,000 X g at room temperature for 10 min.

24. Collect the upper, aqueous phase.

25. Add 7.5 M Ammonium Acetate, pH 7.7 to a final concentration of 2.5 M and 2.5 volume of ice-cold 100% Ethanol to precipitate the DNA.

26. Centrifuge at 10,000 X g for 10 min at 4℃ to pellet the DNA.

27. Wash the DNA pellet with 70% Ethanol.

28. Centrifuge at 10,000 X g for 10 min at 4℃ to pellet the DNA.

29. Resuspend the DNA pellet in 50 μl of TE.

Solutions

Wash Buffer		76% (v/v) Ethanol 10 mM Ammonium Acetate
Chloroform:Isoamyl Alcohol		(24:1) Chloroform:Isoamyl Alcohol (CAUTION! see Hint #6)
Phenol:Chloroform		1:1 Phenol:Chloroform (CAUTION! see Hint #6) Store at 4°C in a dark glass bottle
CTAB Isolation Buffer (see Hint #5)		1.4 M NaCl 100 mM Tris-HCl, pH 8.0 20 mM EDTA 2% (w/v) Ethyltrimethylammonium Bromide (CTAB) 0.2% (v/v) 2-Mercaptoethanol
70% (v/v) Ethanol
7.5 M Ammonium Acetate, pH 7.7
TE		pH 8.0 1 mM EDTA 10 mM Tris-Cl

BioReagents and Chemicals

Isoamyl Alcohol

Chloroform

Phenol

Sodium Chloride

Ethyltrimethylammonium Bromide (CTAB)

RNase A

Tris

Nitrogen, Liquid

Ethanol

2-Mercaptoethanol

EDTA

Ammonium Acetate

Isopropanol

Protocol Hints

1. The Phenol:Chloroform phase may separate with the plant debris, so it may be seen as two layers: debris and an organic phase.

2. The sample may be left at room temperature for several hours to overnight without problems.

3. You can leave the sample at this step for at least two days without problems.

4. Although it still contains RNA, DNA taken at this stage of purification is generally suitable for restriction digestion.

5. For plants containing high polysaccharide levels, i.e. glutinous sap, increase the concentration of CTAB to 3% or higher. For plants with high concentrations of phenolic compounds (i.e. oaks, walnuts) add 1% (w/v) polycinylpyrollidine (PVP-40).

6. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.