S. pombe transformation by lithium acetate, Simple and Fast Method

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. For the standard fresh-culture LiOAc transformation protocol (e.g., for pREP plasmids), see S. pombe transformation by lithium acetate.

Contents

1. Reagents and media
2. Cryopreservation of competent cells
3. Transformation with cryopreserved competent cells
4. Notes

1. Reagents and media

• 30% glycerol in 0.1M LiAc pH4.9
• 50% PEG4000 ( Sigma-Aldrich- 95904)
• minimal medium with low carbon source. Paper uses SLD 0.67% YNB, 0.5% glucose, but we have found that synthetic complete medium with 3% glucose and 0.67% YNB (6.7 g/L) works also.
• 1/2 YE -> 0.25% yeast extract (Apex - 20-255), 1.5% glucose (Acros - 50-99-7)

2. Cryopreservation of competent cells

1. Grow cells in minimal medium with low carbon source overnight at 30°C with shaking (220 rpm) to 10⁷ cells (an OD₅₉₅ of 0.5).
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 1ml ice cold sterilized water.
4. Resuspend cells in ice cold 30% glycerol in 0.1M LiAc pH 4.9 to 10⁹ cells/mL (i.e. 100x smaller volume than the original culture).
5. Make 50µl aliquots and incubate for 30 minutes on ice to allow glycerol to permeate cells, then place cells directly into -80°C freezer.

3. Transformation with cryopreserved competent cells

1. Thaw cells at 30°C in a water bath for 2 minutes.
2. Mix with 5-10ng of DNA (for plasmid transformation) or 1-2µg (for integration) and 145µl 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. The paper states that the following three steps are optional and don’t affect transformation efficiency:
   1. Pellet cells at 1600 g for 3 minutes and remove supernatant by aspiration.
   2. Resuspend in 500µl 1/2 YE Medium (0.25% yeast extract, 1.5% glucose).
   3. Incubate at 30°C for 1 hour with shaking.
6. Spread directly onto minimal selection plates and incubate at 30°C. If using drug selection, plate first onto YES agar plates and allow cells to grow into a lawn (2-5 days) before replica plating onto media with drugs.

4. Notes

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.