Plasmid Isolation from Yeast

Vladimir Svetlov svetlov at oncology.wisc.edu
Thu Jan 8 13:48:03 EST 1998


In article <34B4F11A.4EE7 at bioch.ox.ak.uk>, byudkin at bioch.ox.ak.uk wrote:

> Anybody know of a good kit to isolate plasmids from S. cerevisae?
> 
> (I'm doing 2-hybrid and need to isolate my interactors.)
This is repost of my reply to the same damn question. In case you two don't
talk to each other <G> enjoy:

> Does anyone know a good kit for isolating plasmids from S.cerevisae.  I'm
> doing 2 hybrid screening and need to pull out my interactors.

I do not think you need a kit for that. The protocol is quite simple, just
remember that you can't use yeast for preparative production of plasmids
(especially with two or three different kinds in the same cell as in case
of two-hybrid system) and you'll have to go through E. coli anyway. The
following is my adaptation of the 10 minute-prep.

Rapid (³ten-minute²) isolation of plasmid DNA from yeast  [based: Hoffman
and Winston, Gene 1987 Jan 1; 57(2-3): 267-272)] 

Protocol:   

1. Inoculate a single colony into 5 ml of appropriate complete medium (e.g.
CM ura- for the URA3-based plasmid carrier) and grow with agitation to
OD600=~1.5 (usually overnight).  

2. Pellet cells from 1.5 ml of culture by centrifugation at 8,000 g for 2
min. at room temperature, resuspend in 1 ml of sterile water, and pellet
again. 

3. Resuspend the cell pellet  in 0.2 ml of lysis buffer (2% Triton X-100,
1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA - can be stored for
3-4 months at room temperature w/out loss of efficiency).  Supplement cell
suspension with 0.2 ml of phenol:chloroform:isoamyl alcohol (25:24:1) and
0.3 ml of acid washed glass beads (0.4 mm diameter, Thomas Scientific),
vortex at high speed for 2 min.  

4. Separate the aqueous phase from cell debris, beads, and organic solvents
by centrifugation at 12,000 g for 5 min. at room temperature; 2-5 ul of the
aqueous phase can be used directly to transform 100 ul of RbCl-treated
competent E. coli cells.

NB: using excessive amounts of the aqueous extract in transformation
reaction results in raid loss of transformation efficiency (probably
because of organics carry-over), normal efficiency for 2 u (high copy)
plasmid is 100-200 colonies per ul of aqueous extract and 5-20 colonies per
ul of extract of the CEN-based (single copy) plasmid (for RbCl-treated
DH5alpha cells).

It's quite simple and works really great. If you still wanna kit, I'm sure
there are a lot of people who'd charge you for making a buffer and
packaging glass beads from a big bag into a small one. 
Regards,
V.

-- 
Vladimir Svetlov
McArdle Lab for Cancer Research
Dept. Oncology
UW-Madison
1400 University Ave.
Madison, WI 53706



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