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Can anyone vouch for a protocol to get good gDNA from fungi?


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#1 bjk1985

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Posted 12 February 2013 - 08:40 PM

Tried with the Promega Wizard kit, has worked fine for plant tissue and bacterial cells from past experience, but not mycelia (in all cases material was first ground using mortar/pestle & liquid nitrogen). Then I tried something I thought should work (using proteinase K) but that was even worse for getting DNA. My first step was to use RNase A at 37 C in buffer of 150 mM tris, 25 mM EDTA, and 1% SDS, but that didn't seem to work at all (and this was before proK). Nanodrop data for all was quite good in terms of nucleic acid purity; on the gel I loaded 200-250 ng of each sample.

I don't know why I am getting such terrible results, both ways. Anyone get good DNA from fungus & how do you do it? I'm working with the Diaporthe sp. and Phomopsis sp., btw, which are both Ascomycota.

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#2 bob1

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Posted 13 February 2013 - 12:34 AM

Try a CTAB/PVP protocol, these are good at removing the cell wall components which are usually the problem. A plant based one should work, as they deal with similar problems.

#3 bjk1985

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Posted 16 February 2013 - 09:01 PM

I stll have issues with the DNA not being as bright as it should be (for ~200 ng based on the A260). I think contaminants are inflating this value in the fungi. I am going to try to purify these with some silica spin columns, since some (DNA) is clearly there. Another opton to reduce contamination would be to first isolate the nuclei or at least release the cytoplasmic components but not the DNA. I need to find a protocol for that, though.

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