LB medium and LB agar recipe
Recipes for the three common LB formulations, how much agar to add for plates, and the antibiotic working concentrations to add once it has cooled.
LB (Lysogeny Broth) 1 L
Preparation time about 20 minutes plus autoclaving.
| Component | Formula weight | Amount |
|---|---|---|
| Tryptone | — | 10 g |
| Yeast extract | — | 5 g |
| NaCl (Miller 10 g / Lennox 5 g / Luria 0.5 g) | 58.44 g/mol | 10 g |
| Agar (for plates only) | — | 15 g |
| Deionised water | — | to 1 L |
Procedure
- Dissolve the tryptone, yeast extract and NaCl in about 950 mL of water.
- For plates, add 15 g of agar. Do not attempt to dissolve it — it goes into solution during autoclaving.
- Make up to 1 L and autoclave at 121 °C for 20 minutes.
- For plates, let the bottle cool to about 50 °C before adding antibiotics. Pouring hotter destroys most antibiotics; much cooler and the agar sets in the bottle.
- Pour roughly 25 mL per 100 mm plate. Flame the surface briefly to pop bubbles, and let them dry lid-ajar in a hood for 20 minutes.
What matters here
Antibiotic working concentrations
Ampicillin 100 µg/mL, kanamycin 50 µg/mL, chloramphenicol 25 µg/mL (in ethanol), tetracycline 10 µg/mL (light-sensitive), spectinomycin 50 µg/mL, gentamicin 10 µg/mL. Stocks are normally made at 1000× so you add 1 mL per litre.
Ampicillin plates have a short life
β-lactamase secreted by growing colonies degrades ampicillin locally, which is why satellite colonies appear around a large colony after a long incubation. Use plates within a few weeks and do not over-incubate.
OD₆₀₀ and cell number
An OD₆₀₀ of 1.0 corresponds very roughly to 8 × 10⁸ E. coli per mL, but this is instrument-specific. Calibrate against plate counts if the number matters.
Frequently asked questions
Agar solidified before pouring
Remelt in a microwave in short bursts with the cap loose, swirling between. Watch for boil-over.
Contamination on plates
Usually from insufficient drying or pouring in a draughty area. Dry plates properly and store inverted at 4 °C in a sleeve.
No colonies on a transformation
Check the antibiotic was added below 50 °C, and that you used the right one for your plasmid marker.