[DIYbio] Re: Fungal DNA extraction methods

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