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264 Tikhmyanova et al.4. pYESTrp2 plasmid: forward primer, FP1 can be used, or 5′-GATGTTAACGAT-ACCAGCC-3′ (Invitrogen: Carlsbad, CA, recommended).Reverse primer 5′-GCG TGA ATG TAA GCG TGA C-3′.2.8. Miscellaneous1. Sterile glass beads, 3–4 mm in diameter, no. 3000, Thomas Scientific (Waltham,MA) 5663L19 or Fisher (Waltham, MA) no. 11-312A.2. Sterile glycerol solution for freezing transformants (65% sterile glycerol, 0.1 MMgSO 4, and 25 mM Tris-HCl, pH 8.0).3. Insert grid from a rack of pipet tips (Rainin RT series [Rainin Instrument, LLC,Oakland, CA], 200 µL capacity).4. A metal frogger (e.g., Dankar Scientific, Reading, MA, no. MC48).5. A plastic replicator (Bel-Art Products, Pequannock, NJ, no. 378776-0002, Bel-Blotter, or Fisher no. 1371213).6. 2X Laemmli sample buffer (0.125 M Tris-HCl, 4% [w/v] SDS, 20% [v/v] glycerol,10% [v/v] 2-mercaptoethanol, and 0.002% [w/v] bromophenol blue, pH 6.8).Add 2-mercaptoethanol shortly before use, store at 4°C, and discard if colorbecomes orange.7. Antibody to cI (Invitrogen, Santa-Cruz) and to LexA (Invitrogen).3. Methods3.1. Creating and Assessing the BaitBefore beginning to hunt for an interactor, it is necessary to construct plasmidsthat reliably express the proteins of interest (baits). Baits are expressed asfusions to the λ phage protein cI and/or to the bacterial protein LexA. Theseplasmids are then transformed (22) into a yeast reporter strain to assess the suitabilityof the bait proteins for library screening. Yeast colonies containing baitsare then tested to determine whether the baits are appropriately synthesized, anddo not exhibit self-activation (trigger the transcription of reporter genes on theirown) and are not toxic. For these purposes, they are compared with previouslyestablished controls (Table 1). If all these requirements are not met, there arestrategies that can be used to modify the bait(s) or screening conditions (23).Rapid movement through the characterization steps is recommended beforestarting a library screen, to diminish artifacts and avoid other difficulties.Although plasmids will be retained for extended periods of time in yeast maintainedon stock plates, using freshly transformed colonies for all experiments(