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Bead Beating: A Primer (continued)

APPENDIX C

DNA Isolation from Plants

Isolating DNA from plant homogenates is often complicated by the presence of polysaccharides and polyphenolics.  Homogenization buffers containing CTAB (cetyltrimethylammonium bromide) and PVP (polyvinylpyrrolidone) are commonly used to remove polysaccharides and polyphenolics, respectively.  Polysaccharides will precipitate in solutions containing CTAB and sodium chloride, while PVP binds polyphenolics.

Homogenization Buffer: 2% cetyltrimethylammonium bromide (CTAB), 1.4 M NaCl, 0.1M Tris/HCl, 20 mM EDTA

Isolation Reagents: CTAB precipitation solution (0.5% CTAB, 0.04 M NaCl), chloroform/isoamyl alcohol (ratio of 24:1), 1.2 M NaCl, isopropanol, TE buffer (10 mM Tris, pH 8, 1 mM EDTA)

Procedure

1. Sample can be homogenized cryogenically, dry, or with buffer, depending upon the nature of the sample.  Cryogenic grinding typically yields the largest DNA fragments.  Whether added before or after grinding, use 1 ml homogenization buffer for every 50 mg of tissue. Incubate for 30 minutes at 65°C.
2. Centrifuge the mixture for 10 minutes to pellet plant debris and clarify the solution.  Transfer the supernatant to a new tube.
3. Add an equal volume of chloroform/isoamyl alcohol and mix the contents gently.  Centrifuge for 10 minutes to separate the phases.
4. Transfer the upper phase to a clean tube and add two volumes of CTAB precipitation solution.  Mix and incubate at room temperature for 60 minutes.
5. Centrifuge for 10 minutes at 12,000 g (or high on microfuge).  Decant and dissolve the pellet in 1.2 M NaCl.
6. Extract the solution with an equal volume of chloroform/isoamyl alcohol.  Centrifuge to separate the phases.
7. Transfer the upper aqueous phase to a new tube and add 0.7 volumes of isopropanol.  Incubate at ­20°C for 30 minutes.  Centrifuge at 12,000 g for 20 minutes.
8. Decant and air-dry the pellet.  Resuspend the pellet in TE buffer.