Glycerol stock

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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

E. coli

  • Grow cells overnight in LB
  • Take 0.5ml
  • Add equal volume of 50% sterile Glycerol (in fridge 2)
  • Transfer the cells to a 1.5ml (cryogen)tube
  • Immediately freeze at -80°C
    register the tube in the lists