Any one here with experience using a screening assay for yeast two hybrid?
I am using yeast two hybrid to screen for interactions, however when I plate my cells on the final medium (to check for interaction) I notice two things:
1) a lot of growth (up to 15-20 spots per 96 spots or even more if I incubate longer, I use a pretty short cut off time to be honest)
2) a huge difference between repetitions (I spot each experiment on 2 plates and I repeat it once (starting from scratch, before mating), so in total 2 times 2 plates, the 2 plates from one experiment seem to overlap for 50-75%, however between the plates from repeated experiments I over a very very low overlap).
So I often end up with eg. experiment 1, try 1, 2 plates, 15 spots on plate 1, 20 spots on plate 2 (75% overlap, meaning +-10 spots that are shown on both plates, the other spots are not the same).
The second time (experiment 1, try 2, from scratch): I will have eg. 12 spots on one plate and 18 on the second plate, but they do not overlap with the first experiment!
In the end I often end up having just 1 spot that pops up on the 4 plates.
I have no idea whether this is something I could expect?
The system is known for false positives and for sure 15 spots that grow out of 96 make no sense, but in terms of reproducibility, this seems a bit odd, or not?
I plate them manual , rather than using a robot.
(Often when using a robot they just spot them on 1 plate, 4 times the same "spot", but this seems not good to me since its 4 spots just from 1 "master spot" , I do not really consider this very accurate since I believe you need independent results from 2 "tries").