Difference between revisions of "Synchronized Competent Yeast Cells"
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== Generation -80°C competent yeast cell stocks for chemical transformation == | == 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. | 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 | + | # Prepare 10 ml preculture in [[EMM]]+[[Supplements]] in a 50ml Falkon tube with the lid turned open a bit fixed with tape. |
| − | # Dilute cells in 200 ml EMM+supplements in | + | # Grow the cells by shaking at 32°C for 8–16 hrs. |
| − | # Grow cells overnight until they reach mid-exponential phase (10<sup>7</sup>/ml which yields a total of ~ | + | # Calculate the cell density by using a counting chamber or measuring OD. |
| − | # Take a 1ml aliquot and store on ice/in fridge as a reference for step | + | # 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 10<sup>7</sup> cells/ml. |
| − | # In | + | # Grow cells overnight until they reach mid-exponential phase (10<sup>7</sup>/ml which yields a total of ~1 × 10<sup>9</sup> cells in total; or 2 x 10<sup>9</sup> when using 200ml). |
| − | # Resuspend the cells in | + | # Take a 1ml aliquot and store on ice/in fridge as a reference for step 13. |
| − | # Spin down cells for | + | # 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. |
| − | # Repeat steps | + | # Resuspend the cells in the tube in 25 ml of EMM without nitrogen ([[EMM-N_-_Minimal_Mating_Medium|EMM-N]]). |
| − | # Resuspend cells in 100 ml of [[EMM-N]] | + | # Spin down cells for 3 min at 1800g and remove supernatant. |
| − | # Incubate for 2-3 hrs at 25°C with shaking. | + | # Repeat steps 8 and 9 once more. |
| + | # Resuspend cells in 100 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 (i.e. 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. | # 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. | + | # 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!''' | #:'''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. | # Centrifuge cells for 5 min at 1600g in a 50ml tubes @ 4°C and remove supernatant. | ||
| Line 27: | Line 31: | ||
# 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. | # 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. | # Store aliquots at -80°C immediately. | ||
| − | #: Cells are stored in | + | #: Cells are stored in racks <ins>Pombe 8</ins> or <ins>Pombe 9</ins> in the freezer room |
# Cryopreserved cells can be stored at -80°C for at least 12 months. | # 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 ([https://doi.org/10.1002/yea.1247 Suga & Hatakeyama 2005]) | #: To use, thaw the cells quickly in a 42°C waterbath, which should increase transformation efficiency ([https://doi.org/10.1002/yea.1247 Suga & Hatakeyama 2005]) | ||
[[Category:Protocols]] | [[Category:Protocols]] | ||
Revision as of 18:42, 16 May 2022
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 100 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 (i.e. 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 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 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)
