Potato Germplasm Enhancement Laboratory

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

Small-scale DNA extraction method

The first half of this method is basically similar to that of Doyle and Doyle (1987). The second half is a process of polysaccharide removal.

  1. Collect small pieces of immature leaves. 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 2 ml of 2x CTAB buffer. Grind them with a pestle (150 mm).

  3. Pour the grindate into a 2.0 ml tube up to two thirds from the bottom.

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

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

  6. Centrifuge at a 10,000 rpm for 5 min. Prepare a new 2.0 ml tube containing 900 µl of isopropanol.

  7. Transfer the aqueous portion (upper) using a micropipet (P-1000) to the tube containing isopropanol.

  8. Gently invert the tube several times.

  9. Centrifuge briefly (= Count one second after speed reaches 10,000 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 800 µl 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 10,000 rpm for 3 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 400 µl of TE buffer and 2 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 20 µl of 5M NaCl, and swirl briefly.

  16. Add 147 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 10,000 rpm for 5 min at 4°C.

  19. Decant the solution to a new 2 ml tube, and then fill up the tube with cold AA-ethanol (stored at -20°C).

  20. Mix well and centrifuge the tube at 10,000 rpm for 3 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 800 µl 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 10,000 rpm for 3 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 50 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). (DNA can be stored at 4°C at least for a year without detectable changes. But, if you will not use them for months, store at -20°C for safe)