Smolke:Protocols/Yeast transformation

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

Day 0
Grow an overnight culture of your chosen yeast strain in appropriate media

Day 1

 * 1) In the morning, dilute overnight culture 1:100 (~OD 0.1) into fresh media. Use 5mL fresh media per transformation.
 * 2) Grow the culture to OD 0.4-0.8 (3.5-4.5 hr)
 * 3) *If you're transforming a plasmid, you don't need high efficiency. Just pull the culture whenever it's convenient.
 * 4) 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.
 * 5) Pellet cells at 3-6000g for 5 minutes at 20 °C. Resuspend in 2 mL sterile water per transformation (so 10 mL water for 5 transformations).
 * 6) Repellet. Resuspend in 200 μL sterile water per transformation.
 * 7) Repellet. Resuspend in 200 μL LiAc/TE per transformation.
 * 8) Repellet. Resuspend in 50 μL LiAc/TE per transformation. Add 5 μL SSD per transformation. Aliquot into chilled eppendorf tubes, 55 μL per tube.
 * 9) Add DNA to each tube. 1 μL of a prep is usually more than necessary.
 * 10) Add 300 μL 40% PEG to each tube. Pipet or vortex to resuspend (shouldn't see any streaks after resuspension)
 * 11) Incubate in the spinner at 30 °C for 30 minutes
 * 12) Heat shock in 42 °C water bath for 15 minutes (can be lengthened up to 40 minutes)
 * 13) Spin down, remove PEG
 * 14) If using G418, resuspend in 1 mL YPD, incubate at 30C for 2-3 hr, then repellet
 * 15) Gently resuspend in 100 μL sterile water, plate on appropriate dropout plates

Day 3
Colonies should be ready. Some strains/plasmids may take an extra day.