Synchronized Competent Yeast Cells

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Protocol based on Rodríguez-López et al. 2017 in which EMM-N is used to synchronize cells after which cells are stored following Suga & Hatakeyama 2005.

These cell are used for the Yeast Chemical Transformations protocol.

Generation -80°C competent yeast cell stocks for chemical transformation

Make sure you have enough sterile Erlenmeyer flasks, tubes, sterile EMM-N, EMM, ddH2O and 30% glycerol 0.1M lithium acetate.

This is a protocol for 200ml medium which yields enough cells for 40 to 50 transformation. If cells for fewer transformations are needed feel free to scale down.

  1. Prepare 10 ml preculture in EMM+Supplements in a 50ml Falkon tube with the lid turned open a bit fixed with tape.
  2. Grow the cells by shaking at 32°C for 8–16 hrs.
  3. Calculate the cell density by using a counting chamber or measuring OD.
  4. Dilute cells in 100ml (or for commonly used strains 200 ml and do everything below twice) EMM+supplements in an Erlenmeyer flask (don't fill by more than 15-30%). Use enough cells for inoculum such that after an overnight incubation time you have 107 cells/ml.
  5. Grow cells overnight until they reach mid-exponential phase (107/ml which yields a total of ~1 × 109 cells in total; or 2 x 109 when using 200ml).
  6. Take a 1ml aliquot and store on ice/in fridge as a reference for step 13.
  7. In a 50ml tubes, centrifuge 50ml of the cells for 3 min at 1800g, room temperature (centrifuge in C 01.049), remove supernatant and add the remaining cells to tubes. Spin down again and remove supernatant.
  8. Resuspend the cells in the tube in 25 ml of EMM without nitrogen (EMM-N).
  9. Spin down cells for 3 min at 1800g and remove supernatant.
  10. Repeat steps 8 and 9 once more.
  11. Resuspend cells in 50 ml of EMM-N and incubate in a fresh Erlenmeyer flask. If the strains have auxotrophic markers, add 1/10th of the normal Supplements concentration (so that reduced amount is 10mg/L for adenine and 22.5mg/L for most other supplements).
  12. Incubate for 2-3 hrs at 25°C with shaking in the shaker in room C 01.049.
  13. Check that cells have become smaller and rounder, under light microscope (compare with aliquot from step 3). If not incubate longer.
  14. Place cell culture on ice for 15 min. In the mean time, pre-cool the centrifuge.
    CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!
  15. Centrifuge cells for 5 min at 1600g in a 50ml tubes @ 4°C and remove supernatant.
  16. Resuspend cells in 25ml ice-cold, sterile water.
  17. Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
  18. Repeat steps 16 and 17.
  19. Resuspend cells in 25ml ice-cold, sterile water and count cells using a heamocytometer to obtain concentration.
  20. Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
  21. Resuspend cells in enough of ice-cold, filter-sterilized 30% Glycerol, 0.1M Lithium acetate (pH 4.9; in door fridge 2) to get to a final concentration of 109 cells/ml.
  22. Prepare 50 μl cell aliquots in 1.5 ml sterile Eppendorf tubes, place aliquots on ice for 2 min. Each aliquot is for one transformation.
  23. Store aliquots at -80°C immediately.
    Cells are stored in racks Pombe 8 or Pombe 9 in the freezer room
  24. Cryopreserved cells can be stored at -80°C for at least 12 months.
    To use, thaw the cells quickly in a 42°C waterbath, which should increase transformation efficiency (Suga & Hatakeyama 2005)