Generally applicable technique for preparing fungal DNA (mini-preps)

• Grow mycelium in liquid culture and filtrate. Mycelium can be lyophilized at this stage for optimal yield, but we normally skip this step
• Freeze mycelium in liquid nitrogen; disrupt under under liquid nitrogen using mortar and pestle. At this stage the material may by stored at -70 °C indefinitely.
• Fill the mycelial powder into an Eppendorf cup to ¼ and add 0.8 ml lysis buffer (1% SDS, 10mM Tris-Cl, pH=8.0, 10 mM EDTA). Vortex for 3 min; avoid vortexing from now on
• incubate in a water bath at 65 °C for 10 min
• Add 0.4 ml 1 M KCl, mix and incubate on ice for 30 min
• Centrifuge in a microfuge at top speed for 15 min
• Remove 1 ml of the supernatant into a new Eppendorf tube
• Add 0.25 ml 50% PEG 6000, mix and incubate on ice for 60 min
• Centrifuge in a microfuge at top speed for 15 min
• Remove and discard supernatant with a blue tip; centrifuge again for a few seconds and remove supernatant carefully with a yellow tip
• Dissolve pellet in 0.180 ml sterile water
• Add 20 µl 5 M sodium acetate, pH=5.2, mix, add 0.2 ml cold propanol-2 and incubate for 30 min on ice
• Centrifuge in a microfuge at top speed for 15 min
• Remove supernatant with a blue tip
• Wash once with 0.4 ml 75% propanol-2 (centrifugation for several seconds is enough at this step)
• Remove the supernatant carefully (blue tip/yellow tip-procedure)
• Dry for several minutes in vacuo or simply air-dry
• Dissolve in 50 µl water

The DNA is appropriate for PCR assays and for restriction analysis. 1 µl is normally enough for PCR. We have used this technique successfully for more than a hundred different zygomycetes and many different ascomycetous plant pathogens. The procedure is fool-proof and works without problems in student courses.