Subject: Re: *DNA isolation*

From: [email-masked] (Cedric Gindro)
Date: Wed, 18 Sep 1996 10:09:09 +0200

In article <[email-masked]>, [email-masked]
(ROGER COLLIN) wrote:

> I am starting a project on RAPD analysis of a saprophytic fungus.  I have 
> looked at a number of methods for the isolation of DNA from fungi and would 
> greatly appreciate hearing from anyone who has had experience in this field.  
> Which method is the quickest yet still gives genomic DNA of a quality which 
> can be used for PCR?
> 
> Thanks, Roger Collin

The CTAB technique works very well for filamentous fungi, sclerotia and
dry fruitbodies. 30 seconds in the microwave oven (stop each time boiling
begins) can enhance the extraction. My experience showed that phenol isn't
necessary for filamentous fungi but very usefull for sclerotia. With some
specimens up to 10 repetitions of chloroform extraction are necessary to
eliminate pigments and strange polysaccharides wich precipitate with the
DNA. You can also avoid coprecipitation of polysaccharides by adding 0.6
vol. of isopropanol at -20 °C, shaking gently and centrifuge already after
30 seconds instead of an overnight precipitation in the freezer. Washing
the DNA pellet at least 3 times with 70 percent ethanol (-20°C) is very
important for a good PCR quality.
Finally I recommend you the PCR protocol of Kangfu Yu and K.P. Pauls 1992,
Nucleic Acids Research, vol 20, No. 10, p 2606, to get less random RAPD
patterns...

Cedric Gindro
Institut de Botanique Systematique
et de Geobotanique
Universite de Lausanne, Suisse

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From: [email-masked] (ROGER COLLIN)
Date: Tue, 17 Sep 96 19:38:36 GMT

Thanks, Roger Collin

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From: [email-masked] (John Pitkin)
Date: 17 Sep 1996 23:47:25 GMT

> Which method is the quickest yet still gives genomic DNA of a quality which 

We use a CTAB extraction method for DNA isolation Cochliobolus carbonum. 
This method has also been tested with several other filamentous fungi. We
routinely use DNA isolated this way for PCR.  I posted the technique
several months ago, and it can also be found in  Pitkin et al,
Microbiology v.142 pp.1557-1565.

-- 
John Pitkin
MSU-DOE Plant Research Laboratory
Michigan State University