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getting rid of AD-Vectors in 2-hybrid

Rafael Maldonado rafael at howard.genetics.utah.edu
Fri Nov 17 19:36:36 EST 1995


On 15 Nov 1995 ehrhardt at mpimg-berlin-dahlem.mpg.de wrote:

> Dear netters:
> 
> Is there anyone having a faster or more convenient method for selection of
> AD-vectors from 2-hybrid positive yeast clones than by selection on selective
> media or using transformation of yeast DNA into Leu- deficient HB101 E.coli.
> In my experience the later system suffers from low tranformation efficiency and
> ultra low growth of the HB101 (up to 12days on M9-Leu minimal media).

Things you can do to speed up growth of HB101:

You can select in LB+amp plates first; once you have got colonies, 
replica plate them to MM-leu+pro+thi.

You can add a -Leu mixture to the E coli MM, as it is done in yeast. You 
should include proline and thiamine too.

Thing you can do to increase the transformation eficiency: extract 
plasmid DNA from 10 ml of yest culture using the fast method (phenol and 
glassbead). Concentrate the cells from the 10 ml minimal medium culture 
in a srew-cap ependorff tube. If you have a bead-beater, use it at 3X30 s. 
Precipitate the supernatant, resuspend in 30 ul of TE and electroporate 
with 1 ul to HB101.


Good luck


	Rafa

___________________________________________________________________
                                             |
Rafael Maldonado                             |  La cita ha sido
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Department of Human Genetics		     |  retirada por respeto
University of Utah			     |
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Rafael.Maldonado at genetics.utah.edu           |
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