LARGE-SCALE LEAF DNA EXTRACTION

  1. Funnel lyophilized, ground leaf tissue into a 25 ml capacity Oakridge tube.
  2. Add 10 mL CTAB extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8, 20 mM EDTA pH 8, 1.4 M NaCl) plus 0.1% beta-mercaptoethanol at 50°C for 3 hours. Gently vortex samples to mix.
  3. Spin for 10' at 4000 rpm. Remove 5-8 ml to a fresh tube.
  4. Extract with 5-8 ml 24:1 chloroform/isoamyl alcohol. Vortex thoroughly.
  5. Centrifuge 15' at 4000 rpm.
  6. Remove upper aqueous layer (approximately 8-12 ml) to another tube. Add 2/3 volume isopropanol, mix, and incubate 10' at RT.
  7. Centrifuge at 4000 rpm for 15'.
  8. Pour off isopropanol and add 5 ml 70% ethanol.
  9. Spin at 4000 rpm for 15'. Pour off ethanol and dry for 30' at 80°C.
  10. Depending on pellet size, resuspend DNA in 1-5 ml TE (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8) or dH20. Allow to sit overnight at 4°C to completely dissolve pellet.

Reference
Kang, et al. Plant Molecular Biology Reporter 16: 1-9.