We use a very simple procedure for extraction of DNA from spores for PCR or other uses. Most important, the spore sample must be clean and free of contaminant fungi. This procedure usually is carried out over a 2-3 day period to cull any potentially parasitized or degraded spores. For a typical sample, we start with approximately 1000-1500 spores to have a good sample of genomic DNA at the end of the procedure.

Once a clean preparation is in hand:

1. Spores are transferred to a 2-ml screw-cap vial and water added to capacity.

2. The vial is placed in a mini-beadbeater, which is operated at 4800 rpm for four minutes. This agitation removes surface debris from spores.

3. Vial contents are transferred to a sieve with 45 µm openings and washed once more.

4. Spores are transferred again to a 2-ml screw-cap vial, a drop of sterile distilled water added, 2-3 three millimeter beads and 4-5 one millimeter beads and placed in the beadbeater.

5. The beadbeater again is operated at 4800 rpm for four minutes, during which time the spores disintegrate.

6. DNA is extracted using the Qiagen DNeasy plant kit.

The kit procedure:

Samples are first mechanically disrupted and then chemically lysed. RNA is removed by RNase digestion during lysis. In the spin column procedure, cell debris is removed and samples are filtered and homogenized by centrifugation through a QIAshredder spin column. Buffering conditions are adjusted, precipitating proteins and polysaccharides, and the lysate is loaded onto the DNeasy Plant spin column. During a brief spin, DNA selectively binds to the silica-gel membrane while contaminants pass through. Remaining contaminants and enzyme inhibitors are removed in one or two efficient wash steps. Pure DNA is then eluted in water or low-salt buffer, ready for use.