Difference between revisions of "Synchronized Competent Yeast Cells"
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# Centrifuge for 5 min at 1600g, 4°C and remove supernatant. | # Centrifuge for 5 min at 1600g, 4°C and remove supernatant. | ||
# Repeat steps 12 and 13. | # Repeat steps 12 and 13. | ||
− | # Resuspend cells in 25ml ice-cold, sterile water and | + | # Resuspend cells in 25ml ice-cold, sterile water and [[Determine concentration of yeast|count cells using a heamocytometer]] to obtain concentration. |
# Centrifuge for 5 min at 1600g, 4°C and remove supernatant. | # Centrifuge for 5 min at 1600g, 4°C and remove supernatant. | ||
# Resuspend cells in enough of ice-cold, filter-sterilized [[Lithium Acetate with glycerol|30% Glycerol, 0.1M Lithium acetate]] (pH 4.9; in door fridge 2) to get to a final concentration of 10<sup>9</sup> cells/ml. | # Resuspend cells in enough of ice-cold, filter-sterilized [[Lithium Acetate with glycerol|30% Glycerol, 0.1M Lithium acetate]] (pH 4.9; in door fridge 2) to get to a final concentration of 10<sup>9</sup> cells/ml. |
Revision as of 11:08, 31 January 2019
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.
- Prepare 20 ml preculture in EMM and grow cells by shaking at 32°C for 8–16 hrs.
- 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).
- Take a 1ml aliquot and store on ice/in fridge as a reference for step 10.
- 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.
- Resuspend the cells in each tube in 50 ml of EMM without nitrogen (EMM-N) + supplements.
- Spin down cells for 3min at 1800g and remove supernatant.
- Repeat steps 5 and 6 once more.
- Resuspend cells in 100 ml of EMM-N and transfer to sterile flask.
- Incubate for 2 hrs at 25°C with shaking.
- 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.
- CRITICAL STEP: From here cells should be kept at 4°C, so pre-cool centrifuge etc!
- Centrifuge cells for 5 min at 1600g in two 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 12 and 13.
- 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 columns Pombe 3 and Pombe 4 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)