Hi guys, I am hoping someone could help me with the yeast two-hybrid mating assay procedure.

1. I have constructured an Aedes albopictus cDNA library using Make Your Own "Mate and Plate" Library System (Clontech). I have run all the requirement tests and the library is in good condition (high complexity & high titer). The yeast strain used was Y187.

2. I have constructed a few bait vectors using a few Chikungunya virus genes and confirmed their sequences. The recombinant bait vectors (pGBKT7-CHIKVgenes) are now in yeast strain Y2HGold.

3. Currently I'm trying to mate these Y187-cDNA library and Y2Hgold-bait vector. I have followed the procedure from the Matchmaker Gold Yeast Two-hybrid System (Clontech) for this mating step.

4. The problem I'm facing now is, we get too high yeast growth, a situation which allow us to get single mating colony ONLY if we streak the 10^-4 dilution on the SD/-Trp/-Leu/X/A.

5. Without the 10^-4 dilution, we couldn't get any single colony, but we could see bluish colour in the middle of the plates --> we take this condition as an indicator that there are some blue colonies exist (the protein-protein interactions do happen).

These are the things aI have done for troubleshooting.

--> I have reduced the Aureobasin to 125 ng/mL, instead of 200ng/mL

--> I have used 0.9% NaCl2 for suspension, to replace the 0.5x YDPA

--> I have confirmed the Y2Hgold-bait vector cell number is 1x10^8 cells/mL before mating

--> I have confirmed the gene of interest is in the Y2HGold yeast strain via PCR

I hope someone could help me with the mating procedure, as I don't know what else I could do now to get the single blue colonies.

Thank you in advance :))

Sha

PhD candidate

Monash University Malaysia