Team:Cambridge/Protocols/Glycerol Stocks

Glycerol Stocks

Preparation of glycerol stocks of bacteria allows for long-term storage at -80°C without compromising viability of cells.

Theory

How it works

Practice

• Preparation of glycerol stocks of bacteria

1. Prepare a liquid culture of bacteria in LB with antibiotic for selection. Use 100-fold dilution of an overnight culture of the strain of interest.
2. Incubate cells at 37° for 3-4 hours until the culture reaches the mid-log phase.
3. Transfer around 1ml of the culture into an Eppendorf tube and mix with an appropriate amount of glycerol to get a final concentration of 20%.
4. For high viabilitystore at -80°.

Tip:For precious strains, storage of two stock vials is recommended. Tip:Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. Tip: When recovering a stored strain, it is advisable to check that the antibiotic markers have not been lost by streaking the strain onto an LB-agar plate containing the appropriate antibiotic(s).

Safety

All materials that come into contact with transgenic bacteria must be autoclaved.