1. Prepare cultures of C. graminicola in small Petri dishes containing 10 ml of Difco Potato Dextrose Broth in each dish. Allow to grow under the lights for about 5-7 days at 25-30C. One dish per isolate is usually enough to yield 10-20 mg of DNA. If two or more cultures are combined, the amount of CTAB extraction buffer used in step 5 may have to be increased.
2. Remove the mycelium from the dish using a spatula and transfer to a 15 ml centrifuge. Freeze samples in -80C freezer for at least 30 minutes.
3. Place in the lyophilyzer for about 12-24 hours.
4. Crush mycelium against the side of the 15 ml tube with a glass rod. The glass rod can be rinsed with a squirt bottle of ethanol, wiped off with a Kim-wipe and reused.
5. Add 1.4 ml of CTAB extraction buffer, replace cap and mix well by inverting or gently shaking the tube. Do not vortex.
6. Incubate at 65ÉC for 30 min.
7. Allow to cool for a few minutes, then add 1.4 ml chloroform and mix by hand shaking for 1-2 min.
8. Centrifuge 3000xg for 15 minutes
9. Remove 1000 mL of the upper aqueous phase and place in a 1.5 ml Eppendorf tube. (Note: at this point some species of Colletotrichum will require an extra phenol extraction).
10. Add 700 mL of isopropanol. Invert several times to mix. Do not freeze or refrigerate.
11. Centrifuge 15 minutes at room temperature and remove the liquid phase from the pellet.
12. Rinse the DNA pellet once by adding 500 ml of room temperature 70% ethanol, mixing well, centrifuging for 2 minutes, and removing the liquid phase.
13. Air dry the pellet for 15-20 minutes or dry in a vacuum centrifuge for five minutes.
14. Redissolve the DNA in 50 to 100 ml TE buffer, pH 8.0, and add 2 ml of RNAse A (10mg/ml, boiled). The samples may need to sit in the refrigerator for several hours with occasional mixing to resuspend. Alternatively, incubate at 65C for 10 min.
DNA prepared with this protocol has been used successfully for restriction enzyme digests, ligation, and transformation.
1X CTAB Extraction Buffer - 100 ml
0.7 M NaCl 4.1 g
0.1 M Tris 10 ml of a 1 M Solution, pH 7.5
EDTA 2 ml of a 500 mM Solution
CTAB 1 g
Mix all of the above, add just enough ultrapure water to make up to 100 ml. Store at room temperature. A 1X concentration is for use with lyophilized tissue. Use a 2X concentration for tissue that is still wet.
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