Transforming E.coli with yeast DNA......THANK YOU ALL!!!
EGUARINOS at mvax.cbm.uam.es
Fri Apr 7 04:13:50 EST 1995
Many people replayed my question about transforming E.coli with yeast
DNA. Thank you all.
Here comes a summary of all the suggestions I got (most of them were very
1) You can get the DNA by the Hoffman & Winston method.(when working with
centromeric plasmid be sure to get enough of it)
-Grow yeast in 2 ml O/N under selective pressure for plasmid.
-Spin down in microfuge tube.
-Resuspend in 200 microliters breaking buffer.
2% Triton X-100
100 mM NaCl
10 mM Tris pH 8.0
1 mM EDTA pH 8.0
-Add approximately 200 microliters of glass beads (0.5 mm diameter)
and 200 microliters of phenol:chloroform:isoamyl alcohol.
-Vortex 2 min at highest speed.
-Spin 5 min in micro centrifuge.
2) If you want to transform CaCl or RbCl competent cells it is better to
clean first the DNA with Promega Wizard Mini columns or Geneclean, this would
give you higher efficiency.
3) It seems that electroporation is the way to go, specially for cenbased
vectors as YCp.
To electroporate take 1ul of the supernatant obtained by the previous
method. (If your electroporation arc, you can use less DNA and still get lots
4) There is a method for direct transfer of plasmid DNA from yeast to
E.coli by electroporation that seems to work well with some strains (but I
don´t know if it works with centromere plasmids, nobody has told me about it).
The reference is Nucl.Acid Res, 20/4 p.917 (1992).
THANK YOU ALL.I hope this will help other people like it helped me.
Centro de Biologia Molecular
Universidad Autonoma de Madrid
eguarinos at mvax.cbm.uam.es
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