Yeast plasmid mini-preps

Gotz von Bulow gotz.vonbulow at stjude.org
Fri Sep 29 11:45:48 EST 1995


In article <44gb5p$n9t at griffin.ccc.nottingham.ac.uk>, Simon Dawson
<Simon.Dawson at nott.ac.uk> wrote:

> Hi,
>    Does anybody out there have a nice method for isolating plasmid
> DNA from yeast? I am particularly looking for a method which gives
> reasonable quality plasmid DNA (dosn't need to be ultra-clean) and
> in great enough quantity to allow easy transformation of E.Coli.
>    Thanx in advance for any help on this one.
> 
> 
>                     Simon Dawson.

Here's a method that I use for rescuing shuttle plasmids from yeast. It
works for me!

Take a loopful of yeast and wash into 0.5 ml water in "eppendorf" tube.
Spin down and discard water.
Resuspend yeast in 200 ul 10 mM NaCl, 0.1% SDS in TE buffer.
Add 0.5 vols zirconium or glass beads (I use zirc) and vortex ca. 1 min.
Add 200 ul water-saturated phenol and vortex ca. 1 min.
Spin down and ethanol precipitate the aqueous phase in 300 mM Na acetate.
Wash pellet X2 in 70% ethanol.
Speed vac and dissolve in 30 ul TE or water.

I electroporate 5 ul of this into homemade competent XL1-blue cells and
usually get >100 colonies per plate. I'm sure that other methods of
transformation will work as well.

Good luck, Gotz.

-- 
Gotz von Bulow, PhD                   email: gotz.vonbulow at stjude.org
Dept Experimental Oncology              tel: (901) 495-2712
St Jude Children's Research Hospital    fax: (901) 495-2032
Memphis, TN 38105
                  -----All opinions are my own----



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