Hi there,

As you don't get any blue clones it is crucial to check the functionality of the blue/white assay with the mating positive and negative controls because you can't state at the moment if there is no interaction or if the detection is unefficient.

Dominique Liger Thanks for your response... Actually, after matting I haven't got any colony on the SD/Leu/Trp/x-a-Gal/Aba plates. However matting culture was observed and found 3 lobed structures of the zygote. But after spreading the matted culture I didn't get any colony neither white nor Blue colony.

Hi again, if you don't get anything on SD-Leu-Trp it means the mating has failed (for some reasons the diploids you spotted were not viable).

Hi Tanveer,

On your first question of not enough colonies of the bait clone on SD-trp, have you tried checking for the bait toxicity? In general, if there is no toxicity then few number of clones obtained initially are fine as long as you get good growth later on in liquid SD-trp media.

On your second question, after mating instead of selection on SD/Leu/Trp/x-a-Gal/Aba plates go for selection on triple dropout media (SD-trp-Leu-His). This is a less stringent media and all weak interacting colonies will survive and once the colonies grow on those plates re streak them on SD/Leu/Trp/x-a-Gal/Aba plates. Believe me, then it will grow on high stringent medium!

I had struggled for more than a year to get it right. I had checked library, bait strain everything else but at last I got colonies by this protocol.

All the best, let me know if you make through.

It may be the mating is failed .

With regards.

Dominique Liger and Yehya A. Salih Thanks for your response I will try it again...

Sumesh K M Thanks I will do this also and will let you know.

Thank you dear Tanveer Ahmed ,, You must again it to obtain good results.

With best greetings.