IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

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


Rafael Maldonado                             |  La cita ha sido
room 6160 Eccles Institute of Human Genetics |  
Department of Human Genetics		     |  retirada por respeto
University of Utah			     |
Salt Lake City, Utah 84112. USA.	     |  a la propiedad 
Rafael.Maldonado at genetics.utah.edu           |
Rafael at howard.genetics.utah.edu	             |  intelectual.
Tel: 801-581-4429			     |
Fax: 801-585-3910			     |

More information about the Yeast mailing list

Send comments to us at biosci-help [At] net.bio.net