Before you start
- Use a bottle at most half full so the medium does not boil over during autoclaving.
Reagents and materials
Totals are for 20 plates (+10% extra). Per-plate (10 cm) amounts are in the middle column.
| Reagent | Per plate (10 cm) | Total |
|---|---|---|
| LB agar (molten) | 25 mL | 25 mL |
| Antibiotic stock (1000×) | 25 µL | 25 µL |
Buffers and recipes
LB (Miller) broth and agar per 1 L
| Tryptone | 10 g |
| Yeast extract | 5 g |
| NaCl | 10 g |
| Agar (plates only, 1.5%) | 15 g |
| Ultrapure water | to 1 L |
Autoclave at 121 °C for 15–20 min (liquid cycle). Add heat-labile antibiotics after cooling to about 50–55 °C.
Common working concentrations of antibiotics (E. coli) per reference
| Ampicillin / carbenicillin | 100 µg/mL |
| Kanamycin | 50 µg/mL |
| Chloramphenicol | 25–34 µg/mL |
| Tetracycline | 10–15 µg/mL |
| Spectinomycin | 50–100 µg/mL |
Usually prepared as 1000× stocks; follow plasmid-specific recommendations.
Equipment
- Autoclave
- 55 °C water bath
- Sterile 10 cm Petri dishes
- Bunsen burner or biosafety cabinet
Procedure
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Weigh and dissolve
Dissolve tryptone, yeast extract and NaCl (and agar for plates) in water.
Autoclave
Autoclave with the cap loosened at 121 °C for 15–20 min on a liquid cycle.
Cool
Cool agar to about 50–55 °C in a water bath (warm but comfortable to hold).
Add antibiotic
Add antibiotic from a 1000× stock and swirl gently to avoid bubbles.
Pour
Pour about 20–25 mL per 10 cm plate near a flame or in a cabinet; flame bubbles away quickly.
Set and dry
Let plates set 20–30 min, then dry lids-ajar for a short time or overnight at room temperature.
Store
Store inverted in bags at 4 °C, labelled with antibiotic and date. Use ampicillin plates within about 2–4 weeks.
Critical points
- Antibiotics added to agar that is too hot lose activity.
- Mark plates with a colour code for each antibiotic.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Agar sets before pouring | Keep in the 55 °C bath; pour quickly. |
| Contaminated plates | Pour aseptically; check autoclave cycle. |
| Satellite colonies | Use fresh ampicillin plates or carbenicillin. |
Safety
Autoclave hazards: heat-resistant gloves, never seal bottles tightly.
Further reading
- Green MR, Sambrook J. Molecular Cloning (4th edition): media preparation.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.