Difference between revisions of "Competent Yeast Cells Without Synchronization"
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− | Protocol based on [http://dx.doi.org/10.12688/wellcomeopenres.10038.3 Rodríguez-López et al. 2017] in which EMM-N is used to synchronize cells after which cells are stored following [https://doi.org/10.1002/yea.1247 Suga & Hatakeyama 2005]. | + | Protocol based on [http://dx.doi.org/10.12688/wellcomeopenres.10038.3 Rodríguez-López et al. 2017] in which EMM-N is used to synchronize cells after which cells are stored following [https://doi.org/10.1002/yea.1247 Suga & Hatakeyama 2005]. The protocol omits the EMM-N step as suggested by [https://doi.org/10.12688/wellcomeopenres.16405.1 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. | These cell are used for the [[Yeast Chemical Transformations]] protocol. | ||
− | ==Generation - | + | ==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.'' | ''Make sure you have enough sterile Erlenmeyer flasks, tubes, sterile EMM-N, EMM, ddH2O and 30% glycerol 0.1M lithium acetate.'' | ||
Revision as of 14:25, 27 March 2024
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.
- 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 cells in 25ml ice-cold, sterile water. 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.
- 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 -70°C immediately.
- Cells are stored in racks Pombe 8 or Pombe 9 in the freezer room
- 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)