[Yeast] PCR and sequencing

[Aurélien Boland]

Good morning,
I'm working on a two-hybrid screen and I've 
obtained 109  "positives?" clones that grow 
easily on SD/-TDO medium + x-alpha-gal.
I'm working with S.cerevisiae AH109 yeast (BD 
Matchmaker Library Construction and Screening Kits).
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 indeed,
Have a nice day;

A.BOLAND

Aurélien BOLAND
Cellular and Molecular Research on Plant Biology - URBV
University of Namur - FUNDP
Rue de Bruxelles, 61
B-5000 NAMUR
BELGIUM

Phone: +32 (0) 81/ 72.43.68
Fax: +32 (0) 81/ 72.43.82
e-mail: aurelien.boland at fundp.ac.be
http://www.fundp.ac.be/urbv



    
-------------- next part --------------
An HTML attachment was scrubbed...
URL: http://www.bio.net/bionet/mm/yeast/attachments/20060623/1c091d6e/attachment.html