Difference between revisions of "Glycerol stock"
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== Pombe == | == Pombe == | ||
− | * Grow cells overnight in 5ml liquid medium shaking. | + | * Grow cells overnight in 5ml liquid YES medium shaking. |
* Spin down the cells (or leave overnight in the fridge) | * Spin down the cells (or leave overnight in the fridge) | ||
* Remove supernatant | * Remove supernatant | ||
Line 6: | Line 6: | ||
* Transfer the cells to a 1.5ml (cryogen)tube | * Transfer the cells to a 1.5ml (cryogen)tube | ||
* Immediately freeze at -80°C | * Immediately freeze at -80°C | ||
− | *; register the tube in the lists | + | *; register the tube in the lists for the -80°C |
* alternatively: | * alternatively: |
Revision as of 11:00, 17 April 2020
Pombe
- Grow cells overnight in 5ml liquid YES medium shaking.
- Spin down the cells (or leave overnight in the fridge)
- Remove supernatant
- Resuspend in YES + 15% Glycerol
- Transfer the cells to a 1.5ml (cryogen)tube
- Immediately freeze at -80°C
- register the tube in the lists for the -80°C
- alternatively:
- transfer 0.5ml of liquid culture to a fresh tube
- add 0.5ml 30% or 216 μl 50% sterile Glycerol to the tube
- mix, store and register
Synchronized Competent Yeast Cells should be frozen in Lithium acetate + 30% Glycerol as this increases their transformation efficiency (Suga & Hatakeyama, 2005, doi:10.1002/yea.1247).
Evolution experiment
For the evolution experiment the generations are frozen in 96 well flat bottom plates.
- Spin down cells in the deepwell plates at 1500xg for 3 min.
- Remove supernatant with a 300µl pipette
- add 150µl YES+15%glycerol
- resuspend all cells by pipetting up and down
- with the same tips, move the content to a fresh flat-bottom plate. Make sure you do not touch the side of the wells to avoid cross contamination when opening the plate in the future.
- carefully stick a plastic adhesive film onto the plate.
- replace the lid
- carefully without getting the plastic film contaminated, place the plate in a box and freeze in the -80C freezer