Figure 3. A flowchart for high throughput yeast transformation and random spore analysis of synthetic lethality interactions.

Step 1. Individual haploid-convertible heterozygote diploid YKOs are picked, inoculated onto OmniTrays containing solid YPD plus G418 (200 μg/ml) in a 96-well format, and incubated at 30°C for overnight. Step 2. These strains are inoculated into fresh liquid YPD (100 μl/well) in a shallow 96-well plate with a 96-pin replicator and incubated at 30°C overnight. Step 3. 25 μl of each of the overnight cultures is transferred to a deep 96-well plate containing fresh liquid YPD (1 ml/well). The plate is shaken at 200 rpm at 30°C for 4 hours. Step 3. Cells are pelleted, washed once in 0.1 M LiOAc (200 μl/well), and transferred to a shallow 96-well plate as a cell suspension in 0.1 M LiOAc (100 μl/well). Step 4. After incubation with the transformation mixture at 30°C for 30 minutes and subsequently at 42°C for 13 minutes, cells are pelleted, resuspended in 5 mM CaCl₂ (30 μl/well) for 5–15 minutes, transferred row by row onto solid SC-Ura OmniTray plates, and incubated at 30°C for 2 days. Step 5. Two isolated colonies are picked for each strain and patched onto fresh SC-Ura plates back into the original 96-well format and incubated at 30°C overnight. Step 6. Cells are transferred to solid sporulation medium with a 96-pin replicator and incubated at room temperature (22 to 25°C) for 5 days. Step 7. The sporulated cultures are transferred to a shallow 96-well plate containing sterile water (100 μl/well) with a 96-pin replicator. Cells are resuspended in water by vigorous shaking. Step 8. Two sequential 10 x serial dilutions are made for each row of cell suspensions prepared at the previous step. Step 9. 4 μl of each cell suspension prepared at step 8 is spotted onto solid MM (for xxxΔ::kanMX single and xxxΔ::kanMX yfgΔ::URA3 double mutants), MM-Ura-G418 (for yfgΔ::URA3 single and xxxΔ::kanMX yfgΔ::URA3 double mutants), and MM-Ura (for xxxΔ::kanMX yfgΔ::URA3 double mutants) media and incubated at 30°C for 2–3 days.

Note: For simplicity, this diagram is an abbreviated version of the detailed procedure described in the text and the step numbers may not match those described in the text.