[Yeast] Yeast two hybrid trouble shooting..

Aurélien Boland aurelien.boland at fundp.ac.be
Fri Jun 23 04:20:13 EST 2006


I'm working with the same strain than you but I didn't have this problem...
But I've a problem too... perhaps could find a solution for me or 
find a person that could find it.
I've obtained 109  "positives?" clones that grow easily on SD/-TDO 
medium + x-alpha-gal.
I'm working with S.cerevisiae AH109 yeast.
My problem is that PCR, sequencing (there's nothing, it don't start) 
and even enzymatic restriction don't work...
I've already tried many miniprep protocols before PCR reaction: PCR 
don't work... even colonies screening by PCR don't work... PCR made 
from colonies or miniprep (from bacteria, after transformation either 
by electroporation or thermic choc) don't work too...
PCR on control plasmid that never goes into yeast work fine.
Could you please help me??
Is threre a contminant due to that yeast... Is there modificatin of 
the DNA...???
What could be the problem??
Thank you very much,
Have a nice day;


A. Boland

e-mail: aurelien.boland at fundp.ac.be
http://www.fundp.ac.be/urbv



    
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