quick yeast DNA prep

Michael Lichten lichten at HELIX.NIH.GOV
Fri Mar 24 08:50:05 EST 1995


I cut off the body of the protocol for yeast DNA preparation that was sent
in response to Raymonds request.  The full protocol is reproduced below.

Yeast DNA prep

1.      Grow O/N culture in 5ml YEPD.  Alternatively, grow a confluent
patch on about 1/6 of a plate and resuspend in water.

2.      Spin out cells.  Resuspend in 0.5 ml 1.2 M sorbitol, 0.1 M EDTA, 1%
beta-mercaptoethanol, 1 mg/ml zymolyase, pH 7.5 (important!).  Incubate 30
min at 37C or until spheroplasted (i.e. lyse when put into a drop of 20%
SDS).

3.      Spin briefly (10 sec) in microfuge.

4.      Resuspend pellet in 0.5 ml 100 mM NaCl, 50 mM TRIS, 50 mM EDTA pH 8.=
0.

5.      Add SDS to 1% (i.e. 50 =B5l 10% SDS).  Mix by inverting gently.
Incubate at 65C for 30 min.

6.      Add 0.2 ml 5M KOAc.  Mix by inverting gently.  Incubate on ice, 15 m=
in.

7.      Spin 15-30 min in microfuge at 4=9AC.  Pull off and save supernatant=
.
Avoid carry over precipitate.

8.      Add 0.7 ml isopropanol.  Mix by gentle inversion.  DNA should
precipitate in a large clump.  Let it settle, then decant supernatant.  If
pellet is loose, spin 2 sec in microfuge.  Dry pellet.  Be careful--it will
be slippery.

9.      Resuspend pellet in 0.3 ml TE + 0.3 M NaOAc + 0.1 ug/ml RNase
(boiled).  Incubate at 37C for 30 min.

10.     Add 0.2 ml isopropanol.  Mix by inversion, recover pellet as in
step 8.  Dry pellet, being careful to wipe residual liquid from tube.

11.     Resuspend in 50-100 =B5l TE.  Yield should be 5-10 ugm DNA.

This protocol adopted from the CSH yeast methods book.

=46or an even more rapid protocol that is often sufficient, see

Hoffman CS ; Winston F. A ten-minute DNA preparation from yeast efficiently
releases autonomous plasmids for transformation of Escherichia coli.
Gene 1987;57(2-3):267-72





Michael Lichten
lichten at helix.nih.gov






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