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S.pombe: transformation of cryopreserved competent cells

Protocol taken from paper by Minoru Suga and Toyomasa Hatakeyama “A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved S.pombe cells” Yeast 2005; 22: 799-804

Contents

Section titled “Contents”
  1. Reagents & media
  2. Cryopreservation of competent cells
  3. Transformation of cryopreserved cells
  4. Notes

1. Reagents & media

Section titled “1. Reagents & media”
  • 30% glycerol in 0.1M LiAc pH4.9
  • 50% PEG4000
  • minimal medium with low carbon source (I have been using appropriately supplemented SD with 3% glucose)
  • 1/2 YE (0.25% yeast extract, 1.5% glucose)

2. Cryopreservation of competent cells

Section titled “2. Cryopreservation of competent cells”
  1. Grow cells to 10^7 cells/ml in minimal medium with low carbon source at 30 °C (paper uses SLD 0.67% Bacto yeast nitrogen base, 0.5% glucose).
  2. Place cells on ice for 15 minutes before harvesting.
  3. Pellet cells at 1600 x g for 5 minutes and wash 3 times in ice cold sterilized water.
  4. Resuspend cells in ice cold 30% glycerol in 0.1M LiAc pH 4.9 to 10^9 cells/ml.
  5. Make 50ul aliquots and incubate for 30 minutes on ice to allow glycerol to permeate cells then place directly into -80°C freezer.

3. Transformation of cryopreserved cells

Section titled “3. Transformation of cryopreserved cells”
  1. Thaw cells at 30°C in a water bath for 2 minutes.
  2. Mix with 5-10ng of DNA (sufficient for plasmid transformation but worth adding 1-2ug for integration) and 145ul of 50% PEG4000.
  3. Vortex gently and incubate at 30°C for 1 hour.
  4. Heat shock at 43°C for 15 minutes, allow to cool to room temperature for 10 minutes.
  5. Pellet cells at 1600 x g for 3 minutes and remove supernatant by pipetting.
  6. Resuspend in 500ul ½ YE medium.
  7. Incubate at 30°C for 1 hour with shaking.
  8. Spread directly onto minimal selection plates and incubate at 30°C.

4. Notes

Section titled “4. Notes”

The paper states that steps 5, 6 & 7 can be omitted, plating straight after the 43°C heat shock and cooling steps does not affect transformation efficiency

For plasmid transformation expect several hundred colonies with this protocol. For chromosomal integration I have had varying results, from below 10 to nearly 200 colonies per plate.