Plant Total DNA Isolation
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.
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