Fungal DNA extraction methods

[Brendan Desmond]

Hello everyone,

First time poster, longtime reader. Undergrad biochem major looking to learn more about mycology, microbiology, and synthetic and molecular biology.

I need to obtain DNA from some unknown endophytic fungi that I've been growing on both potato dextrose agar and in potato dextrose broth. My plan is to amplify the DNA I find via PCR and have it sequenced. Some protocols I've obtained from previous research call for a simple method that, in summary, involves crushing, sonicating, and heating fresh mycelia in water or TE buffer. This has been mostly unsuccessful for my colleagues, and I'd prefer to try a more thorough method. However, there seems to be a multitude of methods out there so I'd like to get some opinions.

I found an experiment similar to mine that used the Qiagen DNeasy Plant Mini Kit (http://aem.asm.org/content/77/17/6076), but the smallest set would cost me over two hundred dollars and I simply can't afford it.

A relatively simple method that stuck out to me was described here: http://link.springer.com/article/10.1007%2Fs10327-006-0300-1 , and a link to a PDF is here: https://www.dropbox.com/s/iiae8pg7lsmunle/Simple_Method_for_Fungal_DNA_Extraction_%28sarkosyl%29.pdf?dl=0 .

This procedure involves immersing mycelia in a buffer of Tris/EDTA/NaCl/sarkosyl, centrifugation, and ethanol precipitation. I have access to all components except for sarkosyl, a detergent that is apparently weaker than SDS and does not precipitate at cold temperatures. I am wondering if I can substitute SDS for sarkosyl and centrifuge at room temperature rather than 4 degC.

I'd like to know what people think of this method, or if there are any suggestions for other simple procedures. I am a biochemistry undergrad student and very, very new to fungi, but I'm eager to learn. Does anyone here know of anything I could try, or places I should look?

Thanks and have a good evening.

-Brendan

[Nathan McCorkle]

First I'd check out the 'soil' or 'plant' extraction kits here,
samples are free:
https://mobio.com/technical-support/samples.html

Ethanol precip in that paper doesn't mention that it should be cold or
ice-cold... but the centrifuge steps are at fridge-temp... I don't
think that is cold enough to precip SDS (but you should check). This
method sounds much gentler, which could present total yield problems.
The only thing I'd watch out for is that your centrifuge can get up to
the required level of Gs. (maybe pelleting dirtier DNA is easier with less Gs
because of the contaminants, i.e. with a boiling lysis, etc)

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-Nathan

[Xabier Vázquez Campos]

For fungi there are heaps of DNA extraction protocols, most of them rely on CTAB as surfactant.

I've used the protocol detailed in the paper I attach for high molecular weight DNA for genome sequencing. Hope it helps