Smolke:Protocols/Yeast transformation

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Materials YPD Sterile water Lithium Acetate/TE: 0.1M LiAc, 10mM Tris, 1mM EDTA, pH 7.5 (Mix 1mL 10x LiAc + 1mL 10x TE + 8mL sterile water) 40% PEG: 40% PEG, 0.1M LiAc, 10mM Tris, 1mM EDTA, pH 7.5 (Mix 1mL 10x LiAc + 1mL 10x TE + 8mL 50% PEG) I've seen protocols use different molecular weights of PEG. 3000-4000 seems pretty common Salmon sperm DNA ('SSD', 10 ug/uL, stored at -20C) Plasmid DNA Procedure Day 0 Grow an overnight culture of your chosen yeast strain in appropriate media Day 1 In the morning, dilute overnight culture 1:100 (~OD 0.1) into fresh media. Use 5mL fresh media per transformation. Grow the culture to OD 0.4-0.8 (3.5-4.5 hr) If you're transforming a plasmid, you don't need high efficiency. Just pull the culture whenever it's convenient. Prepare your solutions - put the water, LiAc/TE, and 40% PEG on ice. Thaw the SSD, make an aliquot into a PCR tube, incubate it at 95C for ~5 minutes, then chill on ice. Pellet cells at 6000g for 5 minutes at 4C. Resuspend in 2mL sterile water per transformation (so 10mL water for 5 transformations). Repellet. Resuspend in 200uL sterile water per transformation. Repellet. Resuspend in 200uL LiAc/TE per transformation. Repellet. Resuspend in 50uL LiAc/TE per transformation. Add 5uL SSD per transformation. Aliquot into chilled eppendorf tubes, 55uL per tube. Add DNA to each tube. 1uL of a prep is usually more than necessary. Add 300uL 40% PEG to each tube. Pipet to resuspend (shouldn't see any streaks after resuspension) Incubate in the spinner at 30C for 30 minutes (~2hr if using his3/his5) Heat shock in 42C water bath for 15 minutes Spin down, remove PEG If using G418, resuspend in 1mL YPD, incubate at 30C for ~2hr, then repellet Resuspend in 100uL sterile water, plate on appropriate dropout plates Day 3 Colonies should be ready. Some strains/plasmids may take an extra day. Notes