Mike Barnkob:Protocols/Bacterial/ON liquid culture

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Inoculating overnight liquid culture

Pick colonies and inoculate a liquid culture.

Reagents

Prepare liquid LB media:

  • Following into a 500 ml flask:
    8.2 g LB Broth Ezmix (which includes Tryptone, 5 g/L Yeast Extract, 5 g/L NaCl)
    dH2O up to 400mL
  • Cover flask with aluminum foil, label with autoclave tape and autoclave.
  • Add ampicillin to a concentration of 100 µg/mL (0.4 ml of 100mg/mL)

Protocol

For a single colony:

  1. Add 5 ml of LB + antibiotic to two 50ml Falcon tubes.
  2. Using a sterile loop, select a single colony from LB plate and dip into Falcon Tube.
  3. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
  4. Incubate overnight (12-18 hours) in shaking incubator at 37°C.
    Note: Also incubate a tube that only has LB + antibiotic, but no bacteria as a control. No bacteria should be visible in this after incubation.
  5. Check for growth
  6. Proceed to create a glycerol stock and isolate plasmid DNA.

References

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