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.
Rafael Maldonado | La cita ha sido
room 6160 Eccles Institute of Human Genetics |
Department of Human Genetics | retirada por respeto
University of Utah |
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