I am using a one-hybrid system from Clontech. I have made my dual reporter strain using the HIS3 and LacZ reporters and have transformed with a cDNA-AD library. So, now I would like to do a colony lift b-gal assay on my possible positive colonies. Can anyone tell me how to detect false positives in this assay (those that do not have a bound protein driving expression)? For example, when I tested my untransformed strain for leakiness, the colonies turned blue in 1 hour. Does that mean that when I test my library transformed colonies, that anything that turns blue in more than 1 hour is a false positive? Or is there some other time limit? Is there a reason that an assay using a library transformed colony should take more time than an untransformed colony? And does it make a difference as to how much of the colony was lifted (too much, too little)?
Thanks in advance!
b-gal assay/false positive question
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