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272 Tikhmyanova et al.3.3.1. Transforming the Library1. Use a fresh colony of PRT475 to inoculate a flask containing 20 mL of liquid YPDmedium. Grow yeast overnight at 30°C in an orbital shaker (see Note 4).2. Dilute the overnight culture into 300 mL of fresh YPD liquid medium in a 1-Lflask such that the diluted culture has an OD 600nm of 0.15, and grow as beforeuntil the culture has reached an OD 600nm of 0.5–0.7 (about 4–6 h).3. Subdivide the 300 mL of culture among six 50-mL sterile disposable tubes, andcentrifuge at 1000–1500g for 5 min at room temperature. Gently resuspend eachpellet in 5 mL of sterile water, and combine all the slurries in a single tube. Addsterile water to the top of the tube and mix.4. Recentrifuge the cells at 1000–1500g for 5 min at room temperature. Pour off thewater, add 1.5 mL of TE/0.1 M lithium acetate and resuspend the remaining yeastpellet (~150 µL packed volume). Reserve 150 µL for use in step 10 (parallel controltransformations). Total volume should be approx 1.75 mL.5. Mix 30 µg of library DNA and 1.5 mg of freshly denatured (i.e., boiled for 5 min)sssDNA and mix gently. Add the DNA mix to the yeast. Mix gently and dispense60-µL aliquots of DNA/yeast suspension into 30 microfuge tubes (see Note 15).6. To each tube, add 300 µL of sterile 40% (w/v) polyethylene glycol (PEG) 4000prepared in 0.1 M lithium acetate/TE buffer, pH 7.5. Gently mix by inverting thetubes several times (do not vortex). Incubate the tubes at 30°C for 30–60 min.7. Add 40 µL of dimethyl sulfoxide to each tube, and mix by inversion. Heat-shockcells by placing the tubes in a heat block set to 42°C for approx 10 min.8. Pipet the complete contents of each tube onto each of 30 240 × 240 mm 2 Glu-Trpdropout plate, and spread the cells evenly using 12–24 sterile glass beads. Invertthe plates without discarding glass beads (see Note 16) and incubate at 30°C untilall colonies appear (within 3–4 d).9. Select two representative transformation plates. Draw a 23 × 23 mm 2 (1% of theplate bottom surface) over an area containing an average density of colonies.Count the colonies in each grid section, and recalculate for the whole transformation.A good transformation performed according to this protocol should yieldapprox 20,000–40,000 colonies per plate.10. Use small aliquots (~25 µL) of competent yeast from step 4 to transform the emptylibrary plasmid (pJG4-5 or pYesTrp2), pJG4-5:Raf1, pJG4-5:Krit1, andpYesTrp2:RalGDS. Plate each on a 100-mm plate, and collect the transformedcells as for the library (protocol outlined next), scaling down accordingly.3.3.2. Harvesting and Pooling Primary TransformantsIn the next step, a homogenized slurry is prepared (see Note 17) from thepool of approx 3 × 10 5 –10 6 primary transformants, which is then aliquoted andfrozen. Each of these aliquots is representative of the complete set of primarytransformants, and can be used in subsequent mating.1. Pour 10 mL of sterile water onto each of five 240 × 240 mm 2 plates containingtransformants. Stack the five plates on top of each other. Holding on tightly, shake