>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
>Vienna
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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!
D.Sanglard
Biotechnology ETHZ-Hoenggerberg
CH-8093 Zuerich
sanglard at aeolus.ethz.chsanglard at biotech.biol.ethz.ch
