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Yeast Two Hybrid - (May/20/2007 )

I am using Yeast Strain AH109, pGBKT7 vector for bait, pACT2 vector for library, The library is adult human brain cDNA.

Sequential transformation was used. Bait was firstly transformed into yeast and grows well on SD-T plate. After library transformation, the cells were spread onto 5 SD-T/-L/-H plates. However, only one colony grows on a plate. I have tried to transform only the library into yeast and spread onto SD-L plate, but the yeast grows very well.

Why? is it transformation problem because no library cDNA entered the yeast? or bait protein is toxic? or other problems?



I think you should spread the cells onto 50 SD-T/-L plates after library transformation firstly. Calculate the cotransformation efficiency and collect all the colonies, then spread them onto 5 SD/-T/-L/-H plates.