Synchronized Competent Yeast Cells
From PombEvolution
Revision as of 14:52, 24 May 2022 by Bart (talk | contribs) (→Generation -80°C competent yeast cell stocks for chemical transformation)
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.
- Prepare 10 ml preculture in EMM+Supplements in a 50ml Falkon tube with the lid turned open a bit fixed with tape.
- Grow the cells by shaking at 32°C for 8–16 hrs.
- Calculate the cell density by using a counting chamber or measuring OD.
- 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.
- 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).
- Take a 1ml aliquot and store on ice/in fridge as a reference for step 13.
- 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.
- Resuspend the cells in the tube in 25 ml of EMM without nitrogen (EMM-N).
- Spin down cells for 3 min at 1800g and remove supernatant.
- Repeat steps 8 and 9 once more.
- 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).
- Incubate for 2-3 hrs at 25°C with shaking in the shaker in room C 01.049.
- Check that cells have become smaller and rounder, under light microscope (compare with aliquot from step 3). If not incubate longer.
- 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!
- Centrifuge cells for 5 min at 1600g in a 50ml tubes @ 4°C and remove supernatant.
- Resuspend cells in 25ml ice-cold, sterile water.
- Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
- Repeat steps 16 and 17.
- Resuspend cells in 25ml ice-cold, sterile water and count cells using a heamocytometer to obtain concentration.
- Centrifuge for 5 min at 1600g, 4°C and remove supernatant.
- 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.
- 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.
- Store aliquots at -80°C immediately.
- Cells are stored in racks Pombe 8 or Pombe 9 in the freezer room
- 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)