Cell Preparation (2 h 45 min)
Grow 500 mL cells to desired A600 Centrifuge cellsa for 10 min Add 100 mL YPD/0.02 M HEPES Add 2.5 mL 1.0 M dithiothreitol (DTT) dropwise Incubate for 15 min with shaking at 30°C Add water to 500 mL Centrifuge cells for 10 min Resuspend in 500 mL water Centrifuge cells for 10 min Resuspend in 250 mL water Centrifuge cells for 10 min Resuspend in 20 mL 1.0 M sorbitol Pellet by centrifugation for 10 min Resuspend in 1 mL 1.0 M sorbitol Aliquot into individual 1.5 mL tubes Place in −80°C freezer until needed
Transformation (15 min)
Mix DNA with cells in cuvette Incubate for 2 min on ice Pulse Add recovery medium (optional incubation for 1–3 h) Plate
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Cell Preparation (25 min)
Grow 10 mL cells to desired A600 Centrifuge cellsb for 5 min Resuspend in 10 mL BEDS solution Centrifuge cells for 5 min Resuspend cells in 1 mL BEDS Aliquot into individual 1.5 mL tubes Place in −80°C freezer until needed
Transformation (1 h 45 min)
Mix DNA with cells Add 1.4 mL 40% polyethylene glycol (PEG), 200 mM bicine, pH 8.3 Incubate for 60 min at 30°C Heat shock at 42°C for 10 min (optional recovery for 1–3 h) Pellet cells for 5 min Resuspend cells in 150 mM NaCl, 10 mM bicine, pH 8.3 Repeat steps e and f Plate
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Cell Preparation (30 min)
Grow 50 mL cells to desired A600 Centrifuge cellsb for 5 min Resuspend in 9 mL BEDS + 1 mL 1.0 M dithiothreitol (DTT) Incubate for 5 min with shaking Centrifuge cells for 5 min Resuspend cells in 1 mL BEDS Aliquot into individual 1.5 mL tubes Place in −80°C freezer until needed
Transformation (15 min)
Mix DNA with cells in cuvette Incubate for 2 min on ice Pulse Add recovery medium (optional incubation for 1–3 h) Plate
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