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276 Tikhmyanova et al.the frozen mating, repeat induction and plate at 1 × 10 6 cells per plate on as manyplates as are necessary for full representation of the calculated number of diploids.5. Controls: include the positive control colonies (from mating with the control baitstrains) on each of the master plates. Also, it is appropriate at this time to generateadditional negative controls for subsequent steps by picking at random a fewcolonies from the titer plate (step 1 above) and streaking them in parallel on themaster plates to be tested in the next steps. As these contain randomly chosenlibrary plasmids, the transcriptional activation phenotype of these colonies is mostlikely to be negative: if not, it is necessary to be extremely skeptical of the validityof predicted interactors.6. Incubate the master plates at 30°C until patches/colonies form (overnight).3.3.5. First Confirmation of Positive InteractionsThe following steps test the specificity of positive interactors, assessing the activationof both the auxotrophic and colorimetric reporters in a galactose-dependentfashion. Simultaneously, galactose-inducible activation of both reporters generallyindicate that the transcriptional phenotype is attributable to expression of libraryencodedproteins, rather than derived from mutation of the yeast.1. Invert a replicator, (see Note 7) on a flat surface, and place a master plate upsidedown on the spokes, making sure that the spokes and colonies are properlyaligned. Remove the plate and insert the replicator into a microtiter plate containing50 µL of sterile water in each well. Let the plate sit for 5–10 min, shaking fromtime-to-time to resuspend the cells left on the spokes. When all yeast are resuspended,print on the following plates (see Notes 24 and 25):a. Master plate: Glu/CM Ura − His − Trp − .b. Test for activation of LYS2: Gal-Raff/CM (Ura − His − Trp − ) Lys − and Glu/CM(Ura − His − Trp − ) Lys − .c. Test for activation of LEU2: Gal-Raff/CM (Ura − His − Trp − ) Leu − and Glu/CM(Ura − His − Trp − ) Leu − .d. Two sets of plates, to be assayed for LacZ and GusA activation: Glu/CM Ura −His − Trp − and Gal-Raff/CM Ura − His − Trp − .2. Repeat for each master plate (from Subheading 3.3.4., step 4).3. Incubate the plates at 30°C. After 18–36 h of incubation, take out all Ura − His −Trp − plates. Keep one of the Glu/CM Ura − His − Trp − plates as a fresh masterplate. Overlay the remaining two sets with XGal or XGluc agarose, as describedin Subheading 3.2., step 6. Continue to monitor growth on the Leu − and Lys −plates 48–72 h after plating. For comments on interpretation of the results, referto Table 2.3.3.6. DNA Isolation, and Second Confirmation of Positive InteractionsExecution of the aforementioned protocols for any given bait will result inthe isolation of between zero and hundreds of potential “positive” interactors(see Note 25). These positives must next be evaluated for reproducible phenotype,