Hi everyone,
I have done several Y2H assays by transforming two plasmids (AD and BD derives) into Y187 strains. All of them can grow on the selection medium (SD-LT) medium. However, when I reculture the colonies and transfer them onto SD-LTH and SD-ALTH medium, none of them grow, even with the positive controls. Anyone encounter this kind of situation?
Thanks for your suggestions.
ps: I use the medium from TAKARA, which was bought 5 years ago. I do not think they are expired as they are powder.
Dear Yong,
For me it sound that the proteins you are testing do not interact.
Howeve,r you are writing that even your positive control is not growing?
What is your positive control and what are your growt conditions?
And even if you think that your media should be ok I would check the shelf life - it should be indicated on the product label. And if you don't find anythink I would contact Takara and ask if any problem can occur with the media due to prolonged storage.
These media are designed to only grow yeast strains that demonstrate successful protein synthesis. If your yeast strains are not growing on these media, it is likely due to auxotrophy:
SD-LTH: This medium lacks three essential amino acids: leucine, tryptophan, and histidine.
SD-ALTH: This medium lacks leucine, tryptophan, histidine, and adenine.
For yeast to grow on these media, it must be able to synthesize these amino acids itself.
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