Difference between revisions of "Synchronized Competent Yeast Cells"
From PombEvolution
Jump to navigationJump to searchLine 9: | Line 9: | ||
# Take a 1ml aliquot and store on ice/in fridge as a reference for step 10. | # 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. | # 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 | + | # Resuspend the cells in each tube in 25 ml of EMM without nitrogen ([[EMM-N_-_Minimal_Mating_Medium|EMM-N]]). |
# Spin down cells for 3min at 1800g and remove supernatant. | # Spin down cells for 3min at 1800g and remove supernatant. | ||
# Repeat steps 5 and 6 once more. | # Repeat steps 5 and 6 once more. | ||
− | # Resuspend cells in 100 ml of [[EMM-N]] and transfer to sterile flask. | + | # Resuspend cells in 100 ml of [[EMM-N]] + 1/10th of normal supplements and transfer to sterile flask. |
− | # Incubate for 2 hrs at 25°C with shaking. | + | # Incubate for 2-3 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. | # 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. | ||
#:'''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 | + | # Centrifuge cells for 5 min at 1600g in a 50ml tubes @ 4°C and remove supernatant. |
# Resuspend cells in 25ml ice-cold, sterile water. | # Resuspend cells in 25ml ice-cold, sterile water. | ||
# Centrifuge for 5 min at 1600g, 4°C and remove supernatant. | # Centrifuge for 5 min at 1600g, 4°C and remove supernatant. | ||
Line 26: | Line 26: | ||
# 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 columns <ins>Pombe | + | #: Cells are stored in columns <ins>Pombe 7</ins> or <ins>Pombe 8</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 17:58, 16 November 2021
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 25 ml of EMM without nitrogen (EMM-N).
- 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 + 1/10th of normal supplements and transfer to sterile flask.
- Incubate for 2-3 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 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 columns Pombe 7 or Pombe 8 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)