To begin, obtain the host yeast strain, transformed with the bait plasmid. After introducing the mutant library into it, select a plate with 500 to 2000 colonies. Set two sheets of filter paper on a replica block and make several replicas of the plate, and incubate the plates at 30 degrees Celsius for one to two days.
For the yeast two hybrid color assay, place a sheet of filter paper on the replica plate until it is completely moistened. Remove excess moisture using several sheets of paper towel, twice. Then, using tweezers, peel the filter paper off the plate from the edge and quickly immerse it in liquid nitrogen.
Thaw the frozen filter paper on a dry paper towel with the colony side up. Once thawed, immediately transfer it onto another filter paper soaked with buffer. Cover the plastic tray containing the filter paper, put it into an airtight container, and incubate it at 37 degrees Celsius.
Once blue color appears, place the filter paper on 500 microliters of stop solution taken in a plastic tray and immediately dry it on a fresh paper towel. The color assay produced blue or white dots, differentiating the mutants that did not interact with the bait.