- 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
- 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
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