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Team:Cambridge/Protocols/Glycerol Stocks
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:# 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%. | :# 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%. | ||
:# For high viabilitystore at -80°. | :# For high viabilitystore at -80°. | ||
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| + | '''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 | '''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). | streaking the strain onto an LB-agar plate containing the appropriate antibiotic(s). | ||
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===Safety=== | ===Safety=== | ||
Revision as of 08:08, 21 September 2011
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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
- 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.
- Incubate cells at 37° for 3-4 hours until the culture reaches the mid-log phase.
- 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%.
- 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.
