University of Kentucky
1. Prepare cultures of C. graminicola in 24-well microtiter dishes as follows: Pipette 2 mls of Fries Medium (recipe at the end of this protocol) into each well. Add a small agar plug containing mycelium to each well. Allow to grow under the lights for 4-5 days, shake gently occasionally to disperse conidia. The result should be a mat of mycelium covering the surface of the medium in each well.
2. Transfer the mycelial mats to a stack of clean paper towels using sterile toothpicks. Allow the towels to soak up the liquid from the mats for 10-20 minutes (alternatively, spin the contents of each well in microfuge tubes and pipette off the supernatent). Add 500 microliters of pre-warmed CTAB extraction buffer (recipe at the end of this protocol) to a microfuge tube. Place a mycelial mat in the buffer, and grind the tissue with a sterile mini-pestle as finely and as thoroughly as possible (patience!!). Place each sample as soon as it is ground into a heat block set at 65C. Leave samples at 65C for at least 2 hours.
3. Allow the samples to cool to room temperature, add 500 microliters of chloroform. Roll the samples on the orbital mixer table for 5 minutes, then microfuge for 15 minutes.
5. Remove the upper aqueous phase, and add it to 1 volume of isopropanol. Discard the lower phase in the chloroform waste bottle, tube and all. Rinse the DNA pellet several times in 95% ethanol to remove CTAB.
6. Resuspend the DNA in 200 microliters of TE, and add 1 microliter 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 and 2 volumes of cold 95% ethanol to precipitate the DNA. Spin for 30 minutes in a microfuge, discard supernatent. Resuspend pellet of DNA in 20 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.
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