This protocol describes a method for isolating DNA from plant tissue.

Procedure

1. Preheat the CTAB Isolation Buffer at 60°C.

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°C 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°C.

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°C to pellet the DNA.

27. Wash the DNA pellet with 70% Ethanol.

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

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