Yeast Genomic Preps - Methods or Kits PLEASE!!

Michael Lichten lichten at helix.nih.gov
Mon Sep 22 09:37:46 EST 1997


In article <5vu121$voa at is.bbsrc.ac.uk>, David Hills
<david.hills at bbsrc.ac.uk> wrote:

> Hi,
> does any one out there know of can  recommend a good protocol for genomic 
> extraction of YAC carrying yeast - I don't mind the YACs getting snapped!
> Any body had any experience of kits such as Qiagens' Tips.
> Any help appreciated as I have a *lot* to do.
> Cheers,
> Dave

Here is the method that our lab uses routinely.  It works quite well and
gives excellent yields.  You can scale it down if you only need to do a
few digests.  

We have tried Quiagen tips in the past, and found them not to be worth it,
except if you have a critical sample that for some reason refuses to cut. 
In my opinion, this is almost always due to SDS carry-over from the KAc
precipitation step or due to failure to remove all of the supernatant in
the isopropanol precipitation steps.

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! Zymolyase
doesn't work above pH 8).  Incubate 30 min at 37oC 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 µl 10% SDS).  Mix by inverting gently.  Incubate
at 65oC for 30 min.

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

7. Spin 15-30 min in microfuge at 4oC.  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 µg/ml RNase
(boiled).  Incubate at 37oC 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 µl TE.  Yield should be 5-10 µgm DNA.

This protocol adopted from the CSH yeast methods book.

-- 
Michael Lichten
lichten at helix.nih.gov



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