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Yeast two-hybrid - (Jun/05/2006 )

Hi all, I am using Clontech Yeast two hybrid system. I am screening Arabidopsis flower cDNA library against Zn transporters. I have some serious problem while doing mating experiment. When I do the mating and after that to check the efficiency, I spread some mating mixture in different dilutions to the SD/-Trp (for bait) and SD/-Leu (for library). Everything is according to protocol. The strains that i am using are AH109 and Y187.
But on the plates number of colonies of bait are about 100 fold less than that of library. But accroding to protocol number of colonies should be more in case of bait. Even I am using the # of cells doubled than given in the protocol but getting the same results. Then I do the same mating experiment with the positive controls and got the same results. I don't know what this problem???? it seriously effecting my progress. If anyone knows the soultion plz let me know.


yeast 2 hybrids are evil.

have you checked for contamination. perhaps what you're seeing isn't yeast, but a bacterial infection. we have a guy whose entire project is making a yeast 2 hybrid screen, but he's off sick at the moment.
he had terrible contamination issue a few months back. bacteria had infected one of his stock solutions, and it produced colonies on his plates that looked very similar to yeast. it was not until he took a sample and looked under a microscope, that you could tell is was bacteria.
check your solutions, and if need be, make up a new batch of the broths used. see if there is a yeast guru close at hand, and bother the living daylight out of em.