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Wheat Genetics Resource Center

Kansas State University
Department of Plant Pathology

4024 Throckmorton
1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176 (TEL)
785-532-5692 (FAX)

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383 (TEL)



    1. Collect fresh tissue and store at -20°C.
    2. Grind leaf tissue to a fine powder with liquid nitrogen in a prechilled mortar [Store mortar and pestle at -20°C or -80°C].
    3. Transfer ground tissue with a chilled paintbrush into a well-chilled, 50-ml polypropylene tube and store at -20°C. Do not let the powder thaw. All tools should be chilled in liquid nitrogen.
    4. Add sodium bisulfite to the extraction buffer and adjust pH with 5N NaOH to 7.8–8.0. Heat the extraction buffer to 65°C and add 15–20 ml to 10–15 ml of frozen tissue. Mix well.
DNA extraction buffer
 for 1 L[Final]
5M NaCl
100.0 mL
500 mM
1M Tris-HCl pH 8.0
100.0 mL
100 mM
0.5M EDTA pH 8.0
100.0 mL
50 mM
20% SDS
62.5 mL
0.84% (w/v)
Bring to final volume with sterile distilled water.  Note: Add 0.38 g sodium bisulfite/100 mL extraction buffer just before use and readjust pH to 7.8–8.0 with 5N NaOH. 
  1. Incubate in a 65°C water bath for 30 min; invert the tubes every 5–10 minutes.
  2. Add chloroform:isoamyl alcohol [24:1 v/v] to the top of the tube and mix vigorously.
  3. Centrifuge 15 min at 4,500 rpm. Transfer upper phase into a new 50-ml tube by pouring through 2–4 layers of cheesecloth or pipette off the upper phase if the interphase does not appear solid.
  4. Add 2 volumes (fill tube to top) of cold [-20°C] 95% EtOH and mix gently to precipitate the DNA. Place at -20°C for 30–60 min.
  5. Pour off the 95% EtOH, wash with fresh 70% EtOH, and add 30 ml of cold 70% EtOH. Mix gently for a few minutes. DNA can be left indefinitely in 70% EtOH [can leave overnight at -20°C].
  6. Rewash with fresh 70% EtOH if the DNA is still discolored. Remove DNA by hooking it on a Pasteur pipette and blot off excess 70% EtOH with a Kimwipe. Transfer DNA to the bottom of a sterile 1.5-ml microfuge tube. If making many extractions, centrifuge wet DNA gently, then invert tube a few minutes to let the liquid drain out. Dry DNA at RT for 2–3 hours.
  7. Dissolve DNA in sterile TE [0.5–1 mL depending on pellet size]. Incubate in a 65°C water bath until dissolved with gentle inversion every 30 to 60 minutes or until DNA is dissolved [may take several hours].
  8. Centrifuge 10 minutes in microcentrifuge at 13,000 rpm to remove any material that does not go into solution.
  9. Determine the DNA concentration by separating a 1:20 dilution [1 DNA:20 TE:1 loading buffer solution] on an agarose gel along with markers (uncut lambda DNA at concentrations of 10, 20, 50, and 80 ng). Stain gel with 0.5 μg/ml of ethidium bromide for 15 minutes and photograph. Visually compare the intensity of the DNA bands with the markers to determine the concentration.