Yeast rescue.

S Forsburg forsburg at SALK.EDU
Fri Nov 29 10:38:34 EST 1996


> jjlapres at merle.acns.nwu.edu (John J. La Pres) writes:
> 
> > Here is the scam,  I am currently trying an enhancer trap experiment in which my
> > bait is an enhancer I have isolated from a tissue specific promoter.  I transformed
> > some yeast with this construct and then through a library made from the tissue at
> > this strain.  I then went about rescueing my positive clones.  I have been unable
> > to get any of my transformed bugs to grow. ......


Plasmids from yeast lysates are notoriously hard to transform
into E. coli.  Probably some inhibitors come along.  However,
we have increased our efficiency by several logs by using
ELECTROPORATION to transform our bugs.  It really makes a
huge difference, at least in our hands (yes, from both
pombe and cerevisiae lysates).

So, assuming you saved your lysates....prep up some of your
HB101 to make them electrocompetent, and zap them.  You
won't get a lawn of colonies but you will get enough to work on.

Also, remember that yeast, unlike bugs, can be multiply transformed
 with different plasmids containing the same replicon.  Your 
transformants may carry more than one plasmid, so bear this in mind.

Hope this helps

susan

:;:;:;:;:;:;:;:;:;:;:;:;:;:;:;:;:;:;
S L Forsburg          forsburg at salk.edu
The Salk Institute    http://flosun.salk.edu/~forsburg
La Jolla, CA



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