Glycerol stock

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