Potato Germplasm Enhancement Laboratory

Obihiro Univerisity of Agriculture and Veterinary Medicine
West 2-11, Inada, Obihiro, Hokkaido 080-8555, Japan

Large-scale DNA extraction method

  1. Collect small pieces of immature leaves (young buds are the most preferable).  If soil or chemicals might be attached, wash out with tapping water, and then, excess water must be absorbed by paper towels.  (Matured, well expanded leaves yield less amount of DNA and it may be of low quality)

  2. Place sampled leaves in a mortar (90 mm diameter), and add a pinch of quartz sand and 7 ml of 2x CTAB buffer.  Grind them with a pestle (150 mm).

  3. Pour the grindate into a 30 ml tube as much as possible.

  4. Incubate at 60°C for 30 min with inverting tubes a few times every 10 min.

  5. Add 5 ml of chloroform-isoamyl alcohol to the tube.  Cap tightly and mix by shaking or rocking for 2-3 min.

  6. Centrifuge at 12,000 rpm for 5 min.  Prepare a new 30 ml tube containing 5 ml of isopropanol.

  7. Transfer the aqueous portion (upper) using a diposable 10 ml pipet to the tube containing isopropanol.

  8. Gently invert the tube several times.

  9. Centrifuge briefly (= Count one second after speed reaches over 12,000 rpm, then stop it), decant the solution (DNA is pelleted on the bottom), and hang the tube on a test tube rack to keep the tube upside down.

  10. Wipe out an aqueous drop.  Add 10 ml of 75% ethanol.

  11. Gently invert the tube several times.  Tap the bottom of the tube with a finger to detach DNA pellet from the tube surface into the solution.  Let the tube sit for 30 min (or more).

  12. Centrifuge at 12,000 rpm for 5 min.

  13. Decant the solution, and hang briefly the tube upside down on a test tube rack to drain ethanol solution (1 min).

  14. Add 2 ml of TE buffer and 5 µl of RNase to resuspend DNA pellet and to digest RNA.  Leave the tube until DNA is almost dissolved (approximately 1 hr or more).

  15. Add 100 µl of 5M NaCl, and swirl briefly.

  16. Add 735 µl of absolute ethanol.  Cap the tuber and mix well for a minute.

  17. Cool the tube at 4°C overnight.

  18. Centrifuge the tube at 12,000 rpm for 5 min at 4°C.

  19. Decant the solution to a new 30 ml tube containing 9 ml of cold AA-ethanol (stored at -20°C).

  20. Mix well and centrifuge the tube at 12,000 rpm for 5 min at 4°C.

  21. Decant the solution, and hang briefly the tube upside down on a test tube rack to drain AA-ethanol solution (1 min).

  22. Add 10 ml of 75% ethanol, gently invert the tube several times to detach DNA pellet from the tube surface into the solution and wash it by inverting.

  23. Centrifuge the tube at 12,000 rpm for 5 min at 4°C.

  24. Decant the solution, and hang the tube upside down on a test tube rack to dry the DNA pellet completely (at least 6-8 hrs, or more).

  25. Add 200-300 µl of TE buffer and dissolve the DNA pellet overnight at 4°C.

  26. Centrifuge the tube at 10,000 rpm for a second to collect the DNA solution to the bottom of the tube.

  27. Transfer the DNA solution into a 1.5 ml tube using a P-1000 pipet.

  28. Store in a freezer (-20°C).