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274 Tikhmyanova et al.3. At room temperature, thaw an aliquot of the pretransformed library and negative controllibrary vector strain (see also other controls discussed in step 6). Mix 200 µL ofthe bait strain (~2 × 10 8 cells) with approx 10 8 cells of the pretransformed library(see Subheading 3.3.2., step 4) or negative control strain on a single 100-mmYPD plate and incubate at 30°C for 12–15 h (overnight).4. Add 1.5–2 mL of sterile water and 5–10 (3–4 mm) glass beads to the surface ofeach YPD plate, and suspend the cells as described for library transformation, i.e.,by agitating the plate. Transfer the suspension to a sterile tube and vortex gentlyfor 2 min. Collect the cells by centrifugation at 1000g for 5 min and resuspend inone volume of sterile glycerol solution. Distribute into 200-µL aliquots, and freezeat –80°C (see comment Subheading 3.3.2., step 4). However, leave one aliquotunfrozen if one wishes to proceed directly to the next step—plating on selectivemedium (see Subheading 3.3.4.).5. Titer the mated cells by thawing an aliquot (or using the unfrozen aliquot), andplating serial dilutions (made in sterile water) on Glu/CM Trp − His − Ura − plates(this medium will not support the growth of the parental unmated haploids).Incubate plates at 30°C, and count the colonies that grow after 2–3 d on each plate,and determine the plating efficiency/colony forming units (CFUs) of the matedcells (see Note 20).6. In parallel with steps 1–4 above, grow up approx 1.5 mL of control bait strain(pGLS22-Ras + pLacGus + pEG202-Krev1) in Glu/CM Ura − His − and approx 1.5 mLcultures of each of the three control prey strains (see Subheading 3.3.1., step 10)in Glu/CM Trp − .7. Take a YPD plate and make three spots of control bait strain by placing a drop (~5 µL)of the liquid culture on its surface. Without waiting for the liquid to soak in, overlaywith 5 µL of one of the three control prey strains on each of the spots. Incubateovernight to allow mating, and then streak all three matings onto Glu/CM Ura −His − Trp − plates to select diploids.3.3.4. Screening for Interacting ProteinsIn the next steps, interacting preys are selected by plating the mated cells ontoauxotrophic selection plates. It is important to know how many viable diploidswere plated onto these selection plates both to gain a sense of how much of thelibrary has been screened (saturation) and to determine the false-positive frequency.This information is provided by the titer (expressed as CFU/mL), whichindicates how successful the mating was (see Subheading 3.3.3., step 5).Dual bait reagents allow selection for an interaction with the cI-fused bait(on Gal-Raff/CM Ura − His − Trp − Lys − plates), or the LexA-fused bait (on Gal-Raff/CM Ura − His − Trp − Leu − plates). It also allows selection for preys thatinteract with both baits (on Gal-Raff/CM Ura − His − Trp − Leu − Lys − plates).Negative selection (one interaction but not the second one) is theoretically possible,but impractical in a single step (but very feasible and recommended as afollow-up screen). Hence, one will plate the mated cells (step 2 below) on