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286 Tikhmyanova et al.of tubes. Finally, do not use excess transforming library DNA per aliquot of competentyeast, as competent cells may then take up multiple library plasmids, complicatingsubsequent analysis. Under the conditions described herein, less than 10%of yeast will contain two or more library plasmids.16. Although it is possible to throw away the beads after spreading, it is acceptableand efficient to keep the glass beads on the lids while incubating the plates; glassbeads will be needed to harvest the library transformants (see Subheading3.3.2.). Contamination is much less likely to occur on the glassbeads than on theplates themselves.17. Thoroughly inspect the plates visually before making a slurry to collect transformants.If visible molds or other contaminants are observed on the plates, carefully excise themand a region around them using a sterile razor blade before adding liquid.18. This technique also minimizes the time the plates are open, and thus reduces contaminationfrom airborne molds and bacteria. It is more important to ensure thesame wash-off rate for all plates, than to collect as many yeast as possible (aboutone-third of the yeast slurry will be left on the plates). Furthermore, a significantamount of the water added will soak into the plates, so although 10 mL is added,5 mL is commonly recovered. A second wash can greatly improve the homogeneityof the yield. If one wishes to do a second wash after the first wash, add 10 mL ofwater, shake again, and transfer the slurry to the next unwashed plate; at the end,make sure all yeast are pooled in a common tube. Optionally, the 24 × 24 cm 2plates can be reused many times after removing the remaining agar, washing, andalcohol/ultraviolet sterilization. As these plates are quite expensive, this is a usefulpoint of economy.19. The bait and reporter plasmids should have been transformed into the yeast lessthan about 7–10 d before mating with pretransformed library. If it is older, repeatthe transformation and Western blot.20. Titering can be also be done later, in parallel with selection (see Subheading 3.3.4).21. Compare selection plates seeded with lower and higher densities. The number ofcolonies should be roughly proportional to the seeding density, and there shouldbe no background growth. If disproportionally more colonies (or a lawn) appear onthe more densely seeded plates, this is background resulting from cross-feeding.In this case, a higher number of plates seeded at lower density should be used.Calculate, how many plates at acceptable cell density are necessary for full representationof the desired number of diploids, and if needed, repeat induction andplating from another frozen mating aliquot.22. If colonies do not arise within the first week after plating, colonies appearing atlater time-points are not likely to represent bona fide positives. True interactorstend to come up in a window of time specific for a given bait, with false-positivesclustering at a different time-point: hence, pregrouping by date of growth facilitatesthe decision of which clones to analyze first.23. The number of candidate colonies to pick and characterize should be based on thenumber of cDNA-independent false-positives that arise on the same selectionplates for the control mating. The higher the frequency of false-positives, the more