University of Kentucky
1. Prepare a 2- to 3-day-old shake culture of C. graminicola in Fries Medium (recipe at the end of this protocol). Culture should be shaken at 200 rpm and 30C. Vacuum filter the culture through filter paper in a Buchner funnel, collect the nearly dry mycelium, and weigh it.
2. Grind 2 grams of fresh mycelium in a morter and pestle in liquid Nitrogen. Keep tissue frozen, and grind until the consistency is that of talcum powder.
3. Add 4mls of CTAB extraction buffer (recipe at the end of the protocol) to the powdered tissue, incubate at 65C for 1 hour.
4. Allow the samples to cool to room temperature, add 4mls of chloroform. Roll the samples on the orbital mixer table for 5 minutes, then centrifuge at 6000rpm in the SS-34 rotor for 15 minutes.
5. Remove the upper aqueous phase, and layer it onto 1 volume of isopropanol. Discard the lower phase in the chloroform waste bottle. Remove the DNA glob from the interface of the isopropanol/aqueous mix using a bent glass rod (or spin down, if the DNA won't spool). Rinse the DNA several times in 95% ethanol to remove CTAB.
6. Resuspend the DNA in 1ml of TE, and add 5 microliters of RNAse A (10mg/ml boiled). Incubate at room temperature on the orbital mixer for 30 minutes.
7. Add 1/2 volume of 7.5M ammonium acetate, incubate at room temperature for an additional 30 minutes. This will denature and precipitate proteins. Spin in a microfuge at top speed, transfer supernatent to another tube. Discard pellet.
8. Add 2 volumes of cold 95% ethanol to the supernatent to precipitate the DNA. Spin for 30 minutes in a microfuge, discard supernatent. Resuspend pellet of DNA in 100 microliters of 10 mM Tris pH 7.5 (more or less may be needed, depending on yield).
Fries Medium (1 Liter)
Ammonium tartrate, 5g
Ammonium nitrate, 1g
Potassium phosphate (monobasic), 1g
Magnesium sulfate (7 H2O), 0.48g
Sodium chloride, 1g
Calcium chloride (2 H2O), 0.13g
Yeast extract, 1g
Make up to 1 liter using ultrapure water. Autoclave
CTAB Extraction Buffer,100 mls
20 mls 1M Tris, pH 7.5
28 mls 5M NaCl
4 mls 500mM EDTA, pH 8.0
2 grams CTAB (or CDAB)
Mix all of the above, add just enough ultrapure water to make up to 100 mls. Store at room temperature. Just before using, add b-mercaptoethanol to the volume to be used for the experiment, 20 microliters/ml
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