Preparing genomic DNA of Saccharomyces cerevisiae

Michael Lichten lichten at HELIX.NIH.GOV
Fri Mar 24 08:22:24 EST 1995


>Hello
>
>I'm working with Saccharomyces cerevisiae and I'm looking for a method
>to prepare genomic DNA suitable for restriction enzyme digestion and
>hybridisation with synthetic oligo nucleotides. Is there anybody out
>who can help me? Use the above e-mail adress please.
>
>Many thanks
>  Raymond

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%
=DF-mercaptoethanol, 1 mg/ml zymolyase, pH 7.5 (important!).  Incubate 30 mi=
n
at 37=9AC 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 65=9AC 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 =B5g/ml RNase
(boiled).  Incubate at 37=9AC 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 =B5gm DNA.

This protocol adopted from the CSH yeast methods book.


Michael Lichten
lichten at helix.nih.gov






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