Synchronized Competent Yeast Cells

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

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 20 ml preculture in EMM and grow cells by shaking at 32°C for 8–16 hrs.
2. Dilute cells in 200 ml EMM and grow cells until they reach mid-exponential phase (10⁷/ml which yields a total of ~2 × 10⁹ cells in total).
3. Take a 1ml aliquot and store on ice/in fridge as a reference for step 10.
4. In two 50ml tubes, centrifuge 50ml per tube of the cells for 3 min at 1800g, room temperature, remove supernatant and add the remaining cells to tubes. Spin down again and remove supernatant.
5. Resuspend the cells in each tube in 50 ml of EMM without nitrogen (EMM-N) + supplements.
6. Spin down cells for 3min at 1800g and remove supernatant.
7. Repeat steps 5 and 6 once more.
8. Resuspend cells in 100 ml of EMM-N and transfer to sterile flask.
9. Incubate for 2 hrs at 25°C with shaking.
10. Check that cells have become smaller and rounder, under light microscope (compare with aliquot from step 3). If not incubate longer.
11. Place cell culture on ice for 15 min.

    CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!

12. Centrifuge cells for 5 min at 1600g in two 50ml tubes @ 4°C and remove supernatant.
13. Resuspend cells in 25ml ice-cold, sterile water.
14. Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
15. Repeat steps 12 and 13.
16. Resuspend cells in 25ml ice-cold, sterile water and count cells using a heamocytometer to obtain concentration.
17. Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
18. 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.
19. 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.
20. Store aliquots at -80°C immediately.

    Cells are stored in columns Pombe 3 and Pombe 4 in the freezer room

21. 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)