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Yeast DNA prep; large scale

Dominique Sanglard sanglard at aeolus.ethz.ch
Mon May 24 10:42:59 EST 1993

>I need lots of DNA (1-5 mg per experiment) for studies on oxidative
>damage. Has somebody out there a protocol that works in that scale
>without phenol (or other oxidative damaging steps)?
>DNA can be sheared and contaminated with RNA.
>Thank's very much.
>Christoph Schueller

Dear Christoph,

I worked with the following DNA isolation protocol without the use of
 phenol extraction This prep yields about from 100 to 300 5 microgram
 DNA with a 5 ml culture. I think that the following protocol could be
 scaled up easily to obtain higher DNA amounts.

Isolation of DNA from yeast is performed using 5 ml overnight cultures
 in YEPD.Yeast cells are pelleted by centrifubation 5 min at 5000g,
 washed twice with TE and protoplasted in 2 ml PRO buffer (1 M
 sorbitol, 25 mM ETDA, 20 mM Tris-HCl, pH 7.5) with 0.75 mg
 Zymolyase 100T and 15 mM b-mercaptoethanol. After 30 min
 incubation at 37oC or longer if the protoplasting is not completed, the
 protoplasts are washed twice by centrifugation with the same buffer,
 resuspended in 2 ml lysis buffer (100 mM Tris-HCl pH 8, 50 mM EDTA,
 0.5% SDS, 50 microgram proteinase K per ml) and incubated at 65oC during 30
 min. It is helpful to  resuspended the yeast pellet with a wide bored
 Pasteur pipette to avoid excessive DNA shearing. Proteins are then
 removed by the addition of 1/5 volume of 4 M K-acetate (pH 5) and
 incubation at 4oC during 10 min.  After centrifugation 5 min at 6000g the
 DNA in the supernatant is precipitated with two volumes of ethanol.
 After centrifugation at room temperature during 5 min at 6000 g, the
 pellet is washed with 70% ethanol/water and resuspended in 0.5 ml TE
 buffer. At this step, if desired, this solution can be treated with 20
 microgram RNAse at 37oC during 15 min and DNA re-precipitated in an Eppendorf 
 by the addition of one volume of isopropanol.  A white cocoon is
 formed and can be recovered with a micropipette tip. The resulting DNA
 can be resuspended in a small volume of TE and stored at -20oC before
 digestion with restriction enzymes.

Good luck!

Biotechnology ETHZ-Hoenggerberg
CH-8093 Zuerich
sanglard at aeolus.ethz.ch
sanglard at biotech.biol.ethz.ch

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