Competent Yeast Cells Without Synchronization

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. The protocol omits the EMM-N step as suggested by Torres-Garcia et al. 2020, which reduces the transformation efficiency, but increases the successful integration at the homologous sequence targets.

These cell are used for the Yeast Chemical Transformations protocol.

Generation -70°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. 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.
7. Resuspend cells in 25ml ice-cold, sterile water. CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!
8. Centrifuge cells for 5 min at 1600g in a 50ml tubes @ 4°C and remove supernatant.
9. Resuspend cells in 25ml ice-cold, sterile water.
10. Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
11. Resuspend cells in 25ml ice-cold, sterile water and count cells using a heamocytometer to obtain concentration.
12. Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
13. 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.
14. 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.
15. Store aliquots at -70°C immediately.

    Cells are stored in racks Pombe 8 or Pombe 9 in the freezer room

16. Cryopreserved cells can be stored at -70°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)