[Yeast] No background with yeast two-hybrid screen

Chang, Jennifer via yeast%40net.bio.net (by Jennifer.Chang from nyumc.org)
Thu Jun 18 11:17:58 EST 2009


Hi, All, 

I was hoping that someone may have some insight into my problem as I am
quite stumped. I performed a yeast two-hybrid screen using Clontech's
new Matchmaker Gold system, which tests for interactions by growth on
the antibiotic Aureobasidin A (AbA, 200ng/ml concentration), appearance
of blue color with addition of X-alpha-Gal, growth on -HIS, and growth
on -Ade. My bait was in strain Y2HGold (which apparently is very much
like the strain in their Matchmaker 3 kit - AH109) and my library in
Y187. I thawed an aliquot of my library in Y187 (after thawing, the
titer was ~10^8 cfu/ml) and mated the library strain to the bait strain.
After mating the strains, I tested the viability of the diploids (~10^5
cfu/ml), the bait (~10^8 cfu/ml), and the library (~10^6 cfu/ml) on the
appropriate media and I have screened over 10^6 diploids as the company
manual says to. I initially tested for interactions on the lower
stringency media, which is -LEU, -TRP, X-alpha-Gal, AbA and there was
absolutely no growth. I retested for interactions on lower stringency
medium by reducing the amount of antibiotic to 50ng/ml, 100ng/ml, and
150ng/ml. I still have no growth even on the lowest concentration of
antibiotic. I then tested for interactions on -LEU, -TRP, -HIS,
X-alpha-Gal and I still get nothing! I am really surprised by this last
result as everything I have read suggests that HIS expression is very
leaky and needs to be suppressed by 3-AT in order to detect true
interactions. 

I have previously tested to make sure that my bait does not
auto-activate, is not toxic, and is expressed. The positive and negative
controls provided by the company behave exactly as they should so I know
the media preparation is not the problem. Therefore, I am quite stumped
as to why I am getting absolutely no background, even on media (-HIS)
that is supposed to give high background. Any ideas on what could be
happening? 

Has anyone worked with the Matchmaker Gold kit and what have your
experiences been? 

Best, 
Jennifer 



Regards,
Jennifer Chang, PhD
Post Doctoral Fellow, Cell Biology
Medical Sciences Building, Room 698
New York University School of Medicine
550 First Avenue
New York, NY 10016
Tel 212-263-5316
Fax 212-263-8561 

</PRE>
<html>
<body>
------------------------------------------------------------<br />
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.<br />
=================================
</body>
</html>
<PRE>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: http://www.bio.net/bionet/mm/yeast/attachments/20090618/de51f811/attachment.html


More information about the Yeast mailing list