Transforming E.coli with yeast DNA......THANK YOU ALL!!!

Esther Guarinos EGUARINOS at mvax.cbm.uam.es
Fri Apr 7 04:13:50 EST 1995


  Dear Netters:

     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 
similar).

     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.
  
  	                  Breaking buffer:
	
  		                  2% Triton X-100
  		                  1% SDS
  		                  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.
  
         -Remove supernatant.

     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 
of transformants).
     
     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.


        Esther Guarinos
        Centro de Biologia Molecular
        Universidad Autonoma de Madrid
        eguarinos at mvax.cbm.uam.es
        Madrid   SPAIN




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