Sclerotinia DNA Miniprep

(Based on A. nidulans miniprep: Yelton et al. 1984. PNAS 81:1470-1474)

  1. Add 5-10 small (ca. 2mm2) agar-mycelia plugs to 50ml YPSU media in a 125 ml long neck flask.
  2. Incubate room temperature, 180 rpm, 4-10 days.
  3. Blend cultures with a high-speed handheld mixer; disinfecting with 95% EtOH between cultures.
  4. Harvest mycelia by vacuum filtration through a Buchner funnel lined with Whatman #1 filter paper.
  5. Quick freeze mycelia with liquid nitrogen.
  6. Lyophilize overnight.
  7. Transfer ca. 100 mg of mycelia to an eppendorf tube. Break up the lyophilized mycelia into a fine powder using a tooth pick or metal spatula.
  8. Add 500µl of 50 mM EDTA; 0.2% SDS, pH 8.5. Vortex or mix with tooth pick.
  9. Incubate at 68°C for 10 minutes. Vortex briefly.
  10. Spin 5 minutes in a microfuge (16 000xg).
  11. Transfer supernatant to a new tube and add 30µl 8 M KAc, pH 4.2 (Alkaline Lysis Solution 3:neutralization solution from the original Alkaline Lysis Plasmid Miniprep method. It should have a pH of 4.2-4.8 if prepared as follows: To 60 ml of 5M Potassium Acetate, add 11.5 ml of glacial acetic acid and 28.5 ml of H2O. The resulting sol’n is 3M with respect to potassium and 5M with respect to acetate.) Place on ice for 5 minutes.
  12. Spin 5 minutes in a microfuge.
  13. Transfer supernatant to a new tube and add 600µl isopropanol. Mix well and spin for 5 minutes to pellet DNA.
  14. Invert the tube to dry (or speedvac briefly) and resuspend in 200µl H2O (or TE + RnaseA, 37 C, 30 min)
  15. Add 10µl 10 M LiCl and 500µl 95% ETOH. Mix well and spin for 5 minutes to pellet DNA.
  16. Discard supernantant and dry pellet in speedvac.
  17. Resuspend pellet in 30µl H2O. Determine relative concentration by agarose gel electrophoresis. If a large quantity of RNA is still present, RnaseA treat again and continue with step 18. If little RNA is present and DNA is to be used for Southern analysis, set up digests and run gel.
  18. Phenol:chloroform, then chloroform:IAA extract.
  19. Reprecipitate with LiCl and ETOH.
  20. Wash pellet and speedvac dry.
  21. Resuspend pellet in 30µl H2O.

Updated 19 October 2007 by Jeffrey Rollins.

Commenting is closed for this article.