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Making Glycerol Stocks

From a plate

  • Transfer 0.7 ml sterile growth medium into a 1.5 ml microcentrifuge tube or a cryovial.
  • Add a generous loopful of cell material from the plate and resuspend well.
  • Add 0.3 ml of cold sterile 50% v/v glycerol (this is in the refrigerator) and mix by inversion.
  • Incubate on ice for 30 minutes or more to allow glycerol to penetrate cells.
  • Transfer to the -80 C freezer. In my experience, there is no need to snap-freeze in liquid nitrogen.

From a dense liquid culture

  • Transfer 0.7 ml culture to a 1.5 ml microcentrifuge tube or cryovial.
  • Add 0.3 ml sterile 50% v/v glycerol and proceed as described above.
  • A high density of cells is believed to contribute to cell survival, so if your culture is not very dense, might be a good idea to spin down a larger volume and resuspend the cells in 0.7 ml of medium as the first step.

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