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 l 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 l of competent cells in 1x LiOAc buffer with: *10 l of 10 mg/ml carrier DNA *1-5 g of plasmid DNA or 25% of an efficient PCR (maximum 10 l of total volume).
  3. Add 280 l 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 l of DMSO to each tube. Vortex or pipette to mix thoroughly.
  6. Incubate 5-15 minutes at 42C.
  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 l of TE buffer. For antifungal, resuspend in 200 l of YPD.
  11. Spread cells on appropriate selective plates and incubate for 2-3 days.

Notes

  1. This protocol yields ~500-5000 colonies/g of plasmid DNA. Consider plating high and low volumes of cell suspension (e.g. 160 l and 40 l) 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 -20C 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

1x LiOAc buffer
  • 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 -20C 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 m filter unit.
  5. Store PEG solution in tightly capped container.

-- Main.DaciaLeon - 23 Feb 2017


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Topic revision: r1 - 23 Feb 2017 - 21:16:11 - Main.DaciaLeon