Protocol S2: 96-well yeast transformations Glycerol stocks of the yeast mat




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Protocol S2: 96-well yeast transformations

Glycerol stocks of the yeast MAT haploid deletion strain collection (Open Biosystems, Huntsville, AL) were thawed and pinned onto solid YEPD plates using a 3.18 mm diameter 96 floating pin tool. Before each transformation, a 3.18 mm 96 floating pin tool was used to transfer yeast from the solid media plates to 96-well round bottom plates containing 190 l/well of YPAD media. Plates were incubated overnight at 30C. In the morning, the yeast were resuspended in pre-warmed 2x YPAD media (100 l /well) and the plates were incubated at 30C on platform shaker at 250-300 rpm for 1.5 hours. The yeast were then pelleted and resuspended in 12 l of mix A (0.75l 1M Tris pH 8.0, 0.15 l 0.5M EDTA, 5 l 10 mg/ml boiled salmon sperm DNA , ~ 200ng plasmid DNA and water). Once resuspended, 80 l of mix B (50% PEG3350 + 8.5 l 1M LiOAc) was immediately added to each well. Plates were incubated at 42°C for 105 minutes, the yeast were pelleted and the bulk of the transformation mix was removed. The pellets were resuspended in the residual volume and each well of the 96-well plates was spotted in quadruplicate onto a solid media tray containing synthetic complement minimal media lacking uracil (SC-URA) plus 2% glucose. A 1.58 mm 96 floating pin tool was used for this procedure.


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