- 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
Grow an overnight culture of your chosen yeast strain in appropriate media
- 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 3-6000g for 5 minutes at 20 °C. Resuspend in 2 mL sterile water per transformation (so 10 mL water for 5 transformations).
- Repellet. Resuspend in 200 μL sterile water per transformation.
- Repellet. Resuspend in 200 μL LiAc/TE per transformation.
- Repellet. Resuspend in 50 μL LiAc/TE per transformation. Add 5 μL SSD per transformation. Aliquot into chilled eppendorf tubes, 55 μL per tube.
- Add DNA to each tube. 1 μL of a prep is usually more than necessary.
- Add 300 μL 40% PEG to each tube. Pipet or vortex to resuspend (shouldn't see any streaks after resuspension).
- Incubate in the spinner at 30 °C for 30 minutes.
- Heat shock in 42 °C water bath for 15 minutes (can be lengthened up to 40 minutes).
- Spin down, remove PEG.
- If using G418, resuspend in 1 mL YPD, incubate at 30C for 2-3 hr, then repellet. Alternatively, plate on YPD and let recover overnight, then use replicate plater to replate on selective plates the next day.
- Gently resuspend in 100 μL sterile water, plate on appropriate dropout plates.
Colonies should be ready. Some strains/plasmids may take an extra day.
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 108 cells, 35%w/v PEG, 0.1M LiAc, and 10 μg ssDNA. Cells can be pelleted at 20 °C. Some transformations take 3-4 days for colonies to grow. See http://labs.fhcrc.org/gottschling/Yeast%20Protocols/ytrans.html for more possible variations. ~Stephanie G