Lithium Acetate Transformation

This protocol is originally from the Cold Spring Harbor Laboratory Yeast Genetics & Genomics course manual.

Making Competent _S. cerevisiae Cells

This procedure makes enough competent cells for 5-10 transformations.

1. Grow an overnight culture of each strain in YPD or selective media.
   • The culture should be grown in flasks instead of tubes so the cells are properly aerated.
2. Measure OD600:
   • If OD600 is greater than or equal to 1.0, dilute cells back to 0.1 and grow for 4-6 hours.
   • If OD600 is 0.2-1.0, use immediately or dilute for use later in the day.
   • If OD600 is <0.2, continue growing cells.
3. Transfer the cells to 50 ml conical tubes
4. Pellet cells by centrifugation for 3-5 minutes at 2500 RPM.
5. Remove media and resuspend the cell pellet in 10 ml of 1x LiOAc buffer by vortexing.
6. Centrifuge the cells for 3 minutes at 2500 RPM.
7. Remove liquid and resuspend pellet in 0.5-1.0 ml of 1x LiOAc buffer.
8. Divide into 100 µl aliquots in Eppendorf tubes.
9. Proceed to transformation.

Transforming S. cerevisiae Cells with Lithium Acetate

1. Prepare single stranded carrier DNA.
   • Thaw salmon sperm carrier DNA on ice, boil for 3 minutes, then cool on ice.
2. Combine 100 µl of competent cells in 1x LiOAc buffer with: *10 µl of 10 mg/ml carrier DNA *1-5 µg of plasmid DNA or 25% of an efficient PCR (maximum 10 µl of total volume).
3. Add 280 µl of PEG solution to each tube.
   • Vortex or pipette vigorously to mix the PEG with other components.
4. Incubate 20-45 minutes at room temperature.
5. Add 43 µl of DMSO to each tube. Vortex or pipette to mix thoroughly.
6. Incubate 5-15 minutes at 42°C.
7. Immediately chill on ice for 3 minutes.
8. Centrifuge cells for 2 minutes at half-speed (1250 RPM) and remove liquid.
9. Add 0.5 ml of sterile water, vortex briefly, spin down, and remove liquid.
10. For auxotrophic selection, resuspend in 200 µl of TE buffer. For antifungal, resuspend in 200 µl of YPD.
11. Spread cells on appropriate selective plates and incubate for 2-3 days.

Notes

1. This protocol yields ~500-5000 colonies/µg of plasmid DNA. Consider plating high and low volumes of cell suspension (e.g. 160 µl and 40 µl) to obtain plates with well-spaced transformants.
2. You do not need to boil the carrier DNA every time. Keep a small aliquot in a -20°C freezer and re-boil after three or four freeze/thaw cycles.
3. In our experience, incubating the cells with PEG for 45 minutes leads to a higher transformation efficiency.
4. If cells are incubated at 42 °C for longer than 15 minutes, the cells may go into heat shock resulting in a failed transformation.

Materials

• 0.1 M LiOAc
• 10 mM Tris-HCl (pH 8.0)
• X mM EDTA

1 M LiOAc

1. Dissolve 51 g of LiOAc in 450 ml of water.
2. Adjust volume to 500 ml.
3. Filter sterilize or autoclave.

10x TE

• 10 ml 0.5 M EDTA
• 50 ml 1 M Tris HCl (pH 8.0)
• 440 ml sterile ddH2O

Single-stranded carrier DNA

• 10 mg/ml salmon sperm DNA
• 10 mM Tris-HCl (pH 8.0)
• 1 mM EDTA Boil for 3 minutes, cool on ice, and store at -20°C in aliquots.

PEG solution

1. Heat 50 ml ddH2O in microwave.
2. Add: * 50 g PEG 3350 * 10 ml 10x TE * 10 ml 1 M LiOAc
3. Adjust the volume to 100 ml.
4. Filter sterilize with a 0.45 µm filter unit.
5. Store PEG solution in tightly capped container.

-- Main.DaciaLeon - 23 Feb 2017