I have had this problem before where i was not getting any colonies on the QDO/X/A plates. I figured because they are the most stringent it might be difficult for the yeast to grow on them. Have you tried to change the concentration of Aureubasidin that you put in your plates? I found that using 150 ng/ml (recommended by clontech is 200 ng/ml) was more than enough to select for my positive transformants w/o preventing my positive control from growing.

Regarding the DDO/X, i'm guessing these blue colonies are false positives. Do you use any negative controls other than just the empty plasmids? I know clontech sells a pair of plasmids that are known to not interact in the Y2H system.

Hello, I already tried reducing the Aureubasidin but I'm still getting the same results, anyway thank you!

Probably your candidate prey producing Blue color along with empty vector pGBKT7 is auto-activating or false positive. The reason why you dont get blue color when you use Bait instead of epty vector pGBKT7 is that bait might be masking the binding of prey to the Xgal promoter. Such candidates are known sometimes as systematic false positives. You might like to reverse the fusions of prey and bait to confirm the result.

I agree with Imitaz. You should try to reverse the fusions on the prey and bait plasmids.

Also, what are you using as a positive control? Because if you don't have a solid positive and negative control, it might make the troubleshooting process a little harder.

Imtaz and Toka, I will try the reverse fusions as you suggested, if i do reverse mating (reciprocal interaction), you guys think i can get different results? As for the controls, I still haven't make them, so for sure now I will.

I found from experience that reverse fusions helps with self-activation as well as false negatives. However, this may not be the case with your fusions, nevertheless, I think it's worth a try.

To follow on this question, I would appreciate some input as well. I have constructed a Y2H library (using Clontech's kit) and am now trying to run a screening using my protein of interest. I do get positives on DDO/A but when I patch them on QDO/A, they hardly grow. I already tried to reduced to Aureobasidin concentration to 150, as Toka Omar suggested. I also checked my protein for autoactivation, which it doesn't. Any further advice would be greatly appreciated. Positive control was also fine.

The only thing I noticed is that I do not seem to be able to reach the magic number of 1 million clones screened. Mating efficiency is at about 2.5 % but the number of clones is at around 500k.

Thank you in advance

This answer is many years later but maybe helpful for others.

True interactions would give blue colonies on both DDO/X and QDO/X/A.

Empty bait or no interaction between protein would give white and n/a for the plates.

Sometimes the Gal4 gene is 'leaky' and you might see blue colonies on DDO/X where there isn't a proper interaction.

You might not be seeing colonies on your candidate QDO plate due to a number of reasons. The proteins don't interact. The construct either doesn't fold properly, degrades or occludes the AD-BD interaction.

You could try making a shorter prey or bait by using functional domains. The other option is as mentioned use 150ng of AurA but also perform an outgrowth after transformation in YPDA or YPDA plus before plating.

Hi all, for my Y2H experiment, my yeast gold strain transformed with an empty AD vector (PGADT7) is growing on DDO medium. What could be the possible reasons and how to figure out? Paulina Calderon Flores Lachlan De Hayr Toka Omar Imtiaz Ali

Hello to everyone,

Has someone faced a problem that the positive control pGBKT7-53 + pGADT7-T diploid strain doesn't grow on DDO/X/A. What may be a possible solution to the problem?