Based on Soltis lab protocol from Doyle and Doyle, 1987 and Cullings 1992

  1. Prepare CTAB buffer and add PVP and B-mercaptoethanol.
  2. Weigh out 10-20mg of silica-dried plant tissue or 80-100mg fresh tissue.
  3. Grind tissue.
  4. Add 500uL of CTAB buffer and grind a bit more. Transfer to a labeled 1.5uL tube.
  5. Incubate samples at 55°C for 1hr to overnight. Longer times and periodic shaking give higher yield.
  6. Add 500uL of 24:1 Chloroform:Iso Amyl Alcohol and mix well by shaking tubes by hand for 5 minutes.
  7. Centrifuge for 10-15 minutes at maximum speed. Following centrifugation you should have three layers: the top aqueous phase, the middle debris and proteins, and the bottom chloroform. Go on to the next step quickly so the layers to not remix.
  8. Pipette off the top aqueous layer taking care not to suck up any of the debris or chloroform.
  9. Place the aqueous layer in a new labeled 1.5uL tube. Discard the debris and chloroform.
  10. Estimate the volume of the aqueous layer.
  11. Add 0.08 volumes of cold 7.5M ammonium acetate.
  12. Add 0.54 volumes (using the combined aqueous + ammonium acetate volume) of cold isopropanol (2-propanol).
  13. Mix well by shaking tubes by hand for 5 minutes.
  14. Let sit in the freezer for 15 minutes to overnight. Longer times give higher yield.
  15. Centrifuge for 3 minutes at maximum speed.
  16. Pipette off the liquid, being careful not to lose the pellet with your DNA.
  17. Add 700uL of cold 70% Ethanol and mix by vortexing briefly.
  18. Centrifuge 1 minute at maximum speed.
  19. Pipette off the liquid, being careful not to lose the pellet with your DNA.
  20. Add 700uL of cold 95% Ethanol and mix by vortexing briefly.
  21. Pipette off the liquid, being careful not to lose the pellet with your DNA.
  22. Dry the pellet in the spin-vac for 20 minutes or until all traces (visible or smell) of ethanol are gone.
  23. Resuspend samples with 50-100uL of TE buffer. Allow to resuspend for 1 hour at 55°C or overnight in the refrigerator before quantifying.

Recipes

CTAB buffer (use within 6 months):

  • 25mL 1M pH8 Tris
  • 70mL 5M NaCl
  • 10mL 0.5M EDTA (or 20mL 0.25M EDTA)
  • 5g CTAB
  • water to 250mL
  • Add PVP and B-mercaptoethanol right before starting extractions:
CTAB buffer PVP B-merc
0.5mL 0.02g 2.5uL
5mL 0.2g 25uL
20mL 0.8g 100uL

TE buffer:

  • 5mL 1M pH8 Tris
  • 1mL 0.5M EDTA
  • water to 0.5L

1M pH8 Tris:

  • 121.1g Tris
  • 700mL water
  • Dissolve tris and bring to 900mL
  • pH to 8 with concentrated HCl (will need ~50mL)
  • water to 1L

0.25M pH8 EDTA:

  • 93.06g EDTA
  • 750mL water
  • pH to 8 with NaOH pellets (will need 10-20g. EDTA will not dissolve until pH is near 8)
  • water to 1L

5M NaCl:

  • 292.2g NaCl
  • water to 1L

References:

  • Cullings, K. W. 1992. Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Molecular Ecology 1:233-240.
  • Doyle, J. J. and J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19:11-15.