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 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 I get higher efficiency if I dilute the overnight culture to OD 0.2-0.4 and heat shock for 25 min instead of 15 min. (Gietz & Schiestl's 2007 Nature Protocols paper recommends up to 40 min heat shock, optimized for your strain.) Adjust amounts of PEG (50% w/v), LiAc, and ssDNA so that each transformation has about 10[math]\displaystyle{ ^8 }[/math] cells, 35%w/v PEG, 0.1M LiAc, 0.3mg/mL ssDNA. Cells can be pelleted at 20 °C. Some transformations take 3-4 days for colonies to grow. ~Stephanie