Yeast 2 hybrid Colony lift Assay problem - (May/21/2008 )

Hi All,

I have been trying to perform a yeast-2-hybrid experiment by cloning genes into pGADT7 and pGBKT7 vectors. I use the Y187 strain and detect b-gal activity using colony lift assay. My problem is although the positive control shows blue color within an hour (light blue along the circumference of the colony, not in the center), the interactions i expect to turn blue (which we proved using other methods) do not, even after 8 hrs. This is true for around 4 different genes and their resp. partners.

I am not sure whats happening? are the cells not lysing enough? (i freeze nylon memb in liq. N2 for 10s twice), are the colonies too thick (around 4-5mm dia)?

Any suggestions would be greatly appreciated.

Thanks.

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Why are you using the colony-lift-assay? Does it have some vantages compared to streaking on reporter plates? I streaked the X-alpha-Gal directly on the plates and this worked relatively good (the whole colonies turned blue after some days)
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The reason why i am using Colony lift Assay is bcos i am using the Y187 strain, therefore i need to detect B-gal activity. But i Guess u r using the AH109 strain and thats why u can detect the A-Gal activity by incorporating it into the medium.

Pls do let me know what yeast strain u r using.

Thanks for the earlier comment, and for the new info

You're right I used the AH109 strain.
I was thinking about your question with the colony size and since I made some studies on yeast cell wall I would be carefull with to long grown yeast cells: They rebuild their cell wall and you may get problems with lysis. I would try to use colonies with the size from 1-3mm size and don't let them grow for more then 5 days after Trafo.
Greez jazim