lysis of yeast cells

Rob Kirkpatrick kirkpat at ?cc.umanitoba.ca?
Wed Sep 30 09:03:05 EST 1998


In article <3609213C.1101 at bio.uio.no>, Trygve Meum Eliassen
<t.m.eliasen at bio.uio.no> wrote:

> I am using glass beads (which is the method I have available) to lyse
> S.cer. cells. The method usually works eventually, but it is just so
> inefficient. After 10x1 min of vortex, only a small fraction of the
> cells are lysed. I have collected cells from 50 ml of minimal medium, OD
> 0.5, then added a small volume of phosphate buffer (or SDS-PAGE buffer),
> and glass beads (0.5 mm) to just below the meniscus.  Then 1 min.
> periods of vortex with 1 min. on ice inbetween. I have tried with
> several different tubes; eppendorf and bigger. 
> 
> So obviously I am doing something wrong. I would be grateful if someone
> would tell me what. From thomas.moen at biotek.uio.no


I can't trouble-shoot your method because it is a lot different then ours,
but I'll give you our lysis buffer recipe which we use for glass bead
preps (we call it Yeast Cracking Buffer):

2 ml TritonX-100
5ml 20%SDS
2ml 5M NaCl
2ml 1M Tris-Cl pH8
2ml 0.5M EDTA pH8
bring up volume to 100ml with H2O

We use this to obtain DNA samples for southerns and plasmid rescue in the
Two-hybrid system and it works well.   We start off with a 2ml log-phase
culture of yeast grown in rich culture (may need more volume for minimal
media).  This is pelleted and resuspended in 500microlitres of water and
pelleted again.  This is then resuspended in 200microlitres of our
Cracking Buffer and glass beads are added until a small amount of liquid
is visible.  Next we add 200microlitres of Phenol:Chloroform (1:1).  This
is then vortexed 3 times for 30 sec with at least 30 sec on ice in between
vortexes.  We spin this then remove the upper layer and precipitate it.  I
don't know the actual amount of DNA you get from this but it's plenty for
a southern.

If you're after protein, the story changes.  Let us know exactly what you
want to lyse the cells for and we can give you more specific protocals.

Rob

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