The primary method for long-term E. coli maintenance involves creating glycerol stocks and storing them at ultra-low temperatures. This ensures the viability and genetic stability of your E. coli culture for future use.
Creating Glycerol Stocks for Long-Term Storage
Glycerol stocks are created by mixing E. coli cultures with glycerol, which acts as a cryoprotectant, preventing ice crystal formation that can damage the cells during freezing.
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Grow the E. coli Culture: Grow a fresh, overnight culture of E. coli in a suitable growth medium (e.g., LB broth). Ensure the culture is actively growing and in the exponential phase.
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Prepare Glycerol Solution: Use sterile glycerol to create a final concentration of 30% glycerol in the E. coli culture. This is typically achieved by adding an equal volume of a 60% glycerol solution to the E. coli culture. For example, mix 0.5 mL of a 60% glycerol solution with 0.5 mL of the E. coli culture. It is important to use molecular biology grade glycerol for optimal results.
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Mix Thoroughly: Gently mix the glycerol and E. coli culture to ensure even distribution of the cryoprotectant. Avoid vigorous mixing or vortexing, which can damage the cells.
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Aliquot and Freeze: Transfer the glycerol/E. coli mixture into sterile cryovials. Aliquot into smaller volumes (e.g., 0.5 mL or 1 mL) for easier use and to avoid repeated freeze-thaw cycles of the entire stock. Immediately freeze the vials at -80°C.
Reviving E. coli from Glycerol Stocks
When needed, E. coli can be revived from the glycerol stock:
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Scrape Frozen Stock: Using a sterile loop or pipette tip, lightly scrape the surface of the frozen glycerol stock. Do not thaw the entire vial.
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Inoculate Growth Medium: Transfer the scraped ice crystals into a suitable liquid growth medium (e.g., LB broth) or onto a solid agar plate (e.g., LB agar plate).
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Incubate: Incubate the inoculated medium or plate at the appropriate temperature (typically 37°C) until growth is observed.
Maintaining E. coli During Short-Term Use
For experiments requiring constant access to E. coli cultures, they can be maintained through daily or regular transfers:
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Daily Transfers: Inoculate a fresh medium daily with a small amount of the previous day's culture. This method can introduce mutations over time and is not recommended for long-term maintenance of the strain.
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Regular Transfers: Transfer cultures every few days (e.g., every 2-3 days) and store them at 4°C to slow down growth. This method also increases the risk of mutations over time.
Important Considerations:
- Always use sterile techniques to prevent contamination.
- Label all glycerol stocks clearly with the strain name, date, and any relevant information (e.g., plasmid content, antibiotic resistance).
- Avoid repeated freeze-thaw cycles of glycerol stocks, as this can reduce cell viability.
In summary, creating and properly storing glycerol stocks at -80°C is the most effective method for maintaining E. coli strains long-term, ensuring their availability and genetic integrity for future experiments.
