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yeast one hybrid - beta-gal assay

yeast one hybrid - beta-gal assay - Yeast Forum

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Old 09-30-2008, 09:40 AM
Pipette Filler
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Default yeast one hybrid - beta-gal assay



Dear all,
I am performing a yeast one hybrid screen according the protocol of Deplancke with histidin and beta-galactosidase selection. After screening with a cDNA library, I picked up some colonies on medium lacking histidin + a concentration of 3-AT at which the strain does not self-activate. I retested these positives on a new plate with the same composition: a few colonies could not grow (which seems to me a negative control that is ok), but others again growed well, even a higher concentration of 3-AT... So, it seems to me that there s nothing wrong with this first test.

The problem is that all these colonies don't give blue color after performing the beta-gal assay (protocol according to deplancke, 2006 and clontech: colony-lift filter assay). I tried already multiple times.

Does someone have an idea what could be wrong? The construct containing the LacZ gene is ok: confirmed with PCR, and some strains were blue during testing the self activation in the beginning. Could it be that maybe my not-self activating strain (that i selected for performing the screen) is contaminated with neighboring colonies at my master plate that were self activating, and that the colonies that i picked up after my screen are just self activating colonies instead of positive transformants?

Many thanks in advance...
greetings,
Inge
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Old 06-18-2009, 07:20 PM
Pipette Filler
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Default Re: yeast one hybrid - beta-gal assay

Inge,

Were you able to solve your problem? I am having a somewhat similar one. In my Y2H screen, however, nothing grows. Not even on -HIS, which I am surprised about since it usually needs to be titrated with 3-AT to detect true growth on -HIS/true interactions.

The only thing that I can think of is that the His expression and the B-gal expression are under the control of different promoters and that for some reason the interacting proteins can cause transcription of His but not of B-gal. Or it could be that all the clones you initially picked are revertant mutants for His. That could explain why they can grow on increasing concentrations of 3-AT but don't turn blue. Can you deliberately lose your bait protein plasmid? Once that is lost, the clone should no longer be able to activate His expression. If it can still activate His expression, it would mean you have a revertant.
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assay , beta-galactosidase , betagal , cdna library screen , histidin , hybrid , self acitvation , yeast , yeast-obe-hybrid


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