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:
> 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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