Yeast Electoporation Transformation

Based on Suga et al. 2004 doi:10.2144/04362BM03.

• Grow S. pombe cells in a flask with 50ml-200ml EMM medium with appropriate supplements and 2% glucose overnight to a density of 1 × 10⁷ cells/mL at 32°C.

  don't fill more than 20% of the flask to maintain oxigination! So for larger volumes use a larger flask!

• Place culture on ice for 15 min before harvesting.
• Collect cells by centrifugation at 1800× g for 3 min (centrifuge at 4°C).
• Resuspend the resulting pellet in 5ml ice-cold sterilized water.
• Centrifuge and resuspend 2 more times (so 3 times washing in total).

  Before the last washing, quickly check the concentration of the cells in a haemocytometer.

• Suspend the final pellet in ice-cold 2.0 M Sorbitol to give 5 × 10⁸ cells/mL.
• Make aliquots of 100µl of the cell suspension into 1.5-mL microcentrifuge tubes.
• Freeze these 'slowly' to -80°C (i.e. don't snap-freeze just place in -80°C freezer).

Now the cells can be used for electroporation:

• Thaw the frozen competent cells quickly in a water bath at 30°C.
• Collect cells by centrifugation at 1800× g for 3 min.
• Resuspend the resulting pellet in 1ml ice-cold Sorbitol 1.0M.
• Collect cells again by centrifugation at 1800× g for 3 min.
• Resuspend the resulting pellet in 50µl ice-cold Sorbitol 1.0M (final concentration of 1 × 10⁹ cells/mL).
• Add 1 - 100 ng purified DNA to the mix (keep total volume below 5µl).
• Transfer the cells + DNA mix to a chilled cuvette with 2mm electrode gap.
• Immediately apply a high electric pulse at 2.1 kV, 25 µF, 200 Ω, using the Bio-Rad Gene Pulser II with Pulse Controller Plus (in gel room).

  with 1mm gap use 1000V (says the internet... not tested yet!)

• Add 1 ml Sorbitol 1.0M (room temp) to the electroporated cells and transfer to fresh eppitube.
• Incubate for 30min at 32°C.
• Collect cells by centrifugation at 1800× g for 3 min.
• Resuspend the resulting pellet in 1ml EMM (for homothallic) or EMM-N (for heterothallic strains).
• Incubate at RT overnight or at 32°C for 6 hours.
• Plate out 0.2 ml on YES selection plates.
• Incubate at 32°C. Transformant colonies appeared in 3–6 days.

For old version see: Yeast Electoporation Transformation Old