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glycerol-stocks.md

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Glycerol stocks

TL;DR Store strains in 2 parts media to 1 part 50% glycerol.

MJM collection overview

A strain that is added to the laboratory strain database is assigned an MJM number and stored as a White-capped tube (primary collection) and Yellow-capped tube (backup collection) at -80°C in the designated lab boxes.

Strains that are in progress should be stored as a Red-capped glycerol stocks at -80°C in the lab member's personal box. When they are confirmed to be correct, then they are assigned an MJM number and logged into the strain database. They are then distributed to White- and Yellow-capped tubes and refrozen at -80°C.

When possible, it is a great idea to prepare large numbers of tubes ready to go with glycerol. This improves sterile handling and avoids any barriers to saving important strains. Additionally, the tubes can be autoclaved after glycerol addition to assure sterility.

Large-scale preparation of individual tubes, each containing 50% glycerol

  • Tubes: Wheaton CryoElite 2 ml CS500 cryovials

  • Lay out tubes in a Wheaton rack (VWR 66008-942; bottom of holes are notched to match tubes, so tubes do not move).

  • Unscrew tube caps but leave caps on tubes.

  • Prepare 50% glycerol solution in an autoclaved 250 ml bottle. Add equal volumes of 100% glycerol and nanopure water.

Mix well to distribute the viscous glycerol solution. Using a serological pipet, add the volume listed below to each tube. Slightly close the top on each tube but do not fully seal. Add autoclave tape to each rack and autoclave the racks. After cooling, seal each tube firmly and place on shelf.

  • WHITE (VWR 89125-500): Fill with 300 μl 50% glycerol.
  • YELLOW (VWR 89125-506): Fill with 300 μl 50% glycerol.
  • RED (VWR 89125-502): Fill with 600 μl 50% glycerol.

Freeze White/Yellow-capped glycerol stocks (primary & backup stocks)

  • Complete the lab strain database (see separate protocol).
  • For each strain to be frozen, use two autoclaved freezer vials, each with 300 µl 50% glycerol (on lab media shelf).
  • Label each with the MJM number on the top and side.
  • Add 600 µl cells to each vial of glycerol.
  • Vortex and freeze at -80°C in the designated MJM Strain boxes (White > Primary, Yellow > Backup).

Freeze Red-capped glycerol stocks (tentative stocks)

  • For each strain to be frozen, use one autoclaved freezer vial, which contains 600 µl 50% glycerol (on lab media shelf).
  • Label vial.
  • Add 1200 µl cells to vial of glycerol.
  • Vortex and freeze at -80°C in your personal box.

Transferring Red-capped strains to White-/Yellow-capped vials

  • Complete the lab strain database (see separate protocol).
  • For each strain to be frozen, obtain two empty freezer vials, one White and one Yellow. No additional glycerol should be added.
  • Label each with the MJM number on the top and side.
  • Thaw Red-capped glycerol stock on ice.
  • Once thawed, vortex to mix.
  • Aliquot 900 µl to each of the empty tubes.
  • Freeze at -80°C in the designated MJM Strain boxes (White > Primary, Yellow > Backup).