LB - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Lysogeny-Broth (LB)

This medium is for general growth of E. coli and other bacteria. There are several formulations differing in the amount of NaCl such as LB-Miller (10 g/L NaCl) and LB-Lennox (5 g/L NaCl) and LB Low Salt (≤0.5 g/L NaCl). The higher salt concentrations were originally included for specific experiments with phages and is not necessary for growth of normal E. coli strains. Normal E. coli might grow best on Lennox (5 g/L NaCl). LB Low Salt is useful for salt sensitive antibiotics such as Zeocin or Phleomycin.

LB-Lennox from dry components

  • 10 g/L Tryptone or Peptone
  • 5 g/L yeast extract (very light powder, be careful)
  • 5 g/L NaCl (Lennox), 10 g/L NaCl (Miller) or 0.5 g/L NaCl (Low Salt)
  • Optional: 20 g/L agar for solid medium (*)

Prepare maximum half of the flask volume, so no more than 500 mL in an 1 L flask. This prevents boil over and makes it easier to pour from the bottle. Mix components in a Schott flask, add water and shake well to dissolve all the the powder. Clean the balance if dirty. Clean the spoons and put them on the drying rack. Use the deionized water. Using the tap water in the lab is also ok. We do not check or set pH for this medium. The dry components can also be mixed in a bottle and prepared using a microwave oven, see LBpowder.

(*) Mark the flask with AGAR with big letters on the side of the flask.

Put the flask on the autoclave trolley and fill in the form. Sr. Luis will put the flask in the 50°C incubator to cool down after autoclaving.

Antibiotics

If the media need antibiotics, there are two ways to add it. If all plates need the same antibiotic, it is easiest to add the antibiotics to the medium before pouring the plates. Add antibiotics after cooling down the media bottle to ~50°C (cold enough to touch).

Common antibiotic concentrations:

  • 100 µg/mL Ampicillin (Stock solution is x 1000 concentrated = 100 mg/mL)
  • 50 µg/mL Kanamycin (Stock solution is x 1000 concentrated = 50 mg/mL)

If you need plates with different antibiotics or plates with and without, you can also add the antibiotic to the surface of a solid plate. Add some sterile glass spheres to the plate. Add a puddle of ~200 µL sterile water and about 20 µL of a x 1000 concentrated antibiotic (We assume a plate volume of ~20 mL). Shake the beads to spread and dry under sterile hood if needed.

LB-Luria (50 mL) from stock solutions