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

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 6.7% YNB 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.