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Overnight culture

Overview

2025-12-27_13.12.53.excalidraw|400

|400 (Sezonov et al., 2007)

When E. coli cells are inoculated in 5 mL of LB medium, shaking at ~200 RPM at 37C:

  • Easy carbon source depletion: At OD600 = .3 cell mass starts to decrease, presumably due to the depletion of amino acids that are easy to utilize (Escherichia coli Physiology in Luria-Bertani Broth - PMC). It is also possible that the slow down is influenced by the lack of Mg2+ in the LB medium. If you want to increase Mg2+ availability, use the Mueller-Hinton or MOPS medium (Small Things Considered: The Limitations of LB Medium).
  • Mid-log phase: OD600 = .5 in about 3-4 hours.
  • Stationary phase: OD600 settles down at 2 or 3 in about 8-12 (10^9 cells/mL or .6 mg/mL dry weight).

Materials

  • Liquid LB medium – cold room, 4 mL (glass tubes with blue or black cap)
  • Appropriate antibiotic – -20C fridge by the entrance, a box labelled "antibiotics":
    • Ampicillin: 50 µg/mL
    • Carbenicillin (a more stable ampicillin alternative): 50 µg/mL
    • Chloramphenicol (dissolve in EtOH): 35 µg/mL
    • Kanamycin: 50 µg/mL
  • Bunsen burner
  • Plate with colonies
  • Toothpick
  • 2 ml Eppendorfs – 2x

Procedure

All steps are done behind a Bunsen burner (10 cm behind the flame).

  1. Add 4 uL of antibiotic to the 4 mL of LB medium (i.e., ratio 1:1).
  2. Using a pipette tip gently touch a colony on a plate.
    • Even the slightest of touches will already contain enough colonies. You don't have to see the colony on the tip.
    • If using a frozen culture (from -70C), place the tube with the culture on ice, dip a loop / tip, then return the culture to -70C. For sensitive applications (e.g., competent cell preparation), best practice is to first inoculate on a plate and pick a single colony for the overnight culture.
    • Do not use the recovery from a transformation to immediately grow overnight culture. The transformation output contains a heterogenous population of plasmids and contaminants; plating recovery helps to get rid off them and focus on a single colony.
  3. Scrape off the tip of the pipette into the LB medium tube.
  4. Place in a shaker at 37C for 12-16 hours.
    • Keep the cap loose for aeration.
  5. In the morning, the medium should look cloudy. Divide the liquid into:
    1. 500 uL, add 250 uL glycerol and 250 uL water (i.e., 25% glycerol in the end), and store in -70C for later usage.
    2. 1.6 mL in two 2 mL Eppendorfs and spin at 17000 g briefly (<1 min).
  6. Discard supernatant.
  7. Proceed to plasmid purification. Expected yield: about 100 ng/uL.