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Short answer
The usual ampicillin working concentration for selecting plasmid-carrying bacteria in LB broth or on LB agar plates is 100 µg/mL. Addgene’s plate protocol lists 100 µg/mL, and one manufacturer states that typical working concentrations are 50-100 µg/mL in culture media. From a 100 mg/mL stock, 100 µg/mL means adding 1 µL of stock per mL of medium, which is 100 µL per 100 mL of LB.
This page is about antibiotic selection of bacterial cultures in the laboratory. It is not about drug dosing for patients.
Definition and scope
A working concentration is the final concentration of an antibiotic in the medium the cells actually grow in, as opposed to the concentrated stock solution it is diluted from. For ampicillin, the working concentration is the figure that matters when LB broth or LB agar is used to select bacteria that carry an ampicillin-resistance plasmid, such as those handled in gene cloning workflows and described in a plasmid map.
Two units are easy to confuse. Stocks are usually written in mg/mL and working concentrations in µg/mL, and 1 mg/mL is 1,000 µg/mL. A 100 mg/mL stock is therefore 100,000 µg/mL.
How much ampicillin to add to LB broth
Take the stock concentration, divide it by the working concentration you want, and that is the dilution factor. The table below applies that arithmetic to a 100 mg/mL stock. The two working concentrations come from the sources listed at the end of the page; the dilution figures are our own calculation from them.
| Target working concentration | Dilution from 100 mg/mL stock | Stock per 100 mL of LB | Stock per 1 L of LB |
|---|---|---|---|
| 100 µg/mL | 1:1000 | 100 µL | 1 mL |
| 50 µg/mL | 1:2000 | 50 µL | 0.5 mL |
The same arithmetic works for any stock. For a 50 mg/mL stock, 100 µg/mL is a 1:500 dilution, so 200 µL per 100 mL of medium.
Ampicillin concentration in LB broth versus LB agar plates
Addgene’s protocol for pouring LB agar plates lists a 100 mg/mL ampicillin stock and a 100 µg/mL working concentration, the figure this page uses for broth as well. For plates, the timing of the addition matters. The antibiotic goes into the molten agar only after it has cooled in a 60 °C water bath for at least 5 minutes, so that it is warm to the touch rather than hot. Plates made this way are stored at 4 °C and, per the same protocol, kept no longer than one month.
The manufacturer page says to add ampicillin to sterile, cooled media and not to autoclave it in the medium. For LB broth, that means adding it after the medium has been sterilized and has cooled.
Stock preparation and storage
Addgene’s protocol notes that, unless otherwise indicated, the antibiotic powder can be dissolved in dH2O. Its example is 100 mg of powder in 1 mL of water, mixed by vortexing and then filter sterilized. This is routine media preparation of the kind covered in the buffer and solution preparation guide.
For storage, a manufacturer of a ready-made 100 mg/mL aqueous ampicillin sodium salt solution states that it should be kept at -20 °C and protected from repeated freeze-thaw cycles and from prolonged high temperature. It gives that product a shelf life of 365 days; that figure belongs to that product and is not a general rule for home-made stocks. The Addgene page does not specify a stock storage temperature.
Where the stated concentrations differ
| Source | What it states |
|---|---|
| Addgene, “Pouring LB Agar Plates” | Ampicillin stock 100 mg/mL; working concentration 100 µg/mL |
| Teknova, ampicillin 100 mg/mL product page (manufacturer statement) | Typical working concentrations 50-100 µg/mL in culture media; do not autoclave in media; store at -20 °C |
The two sources do not conflict. One gives the value a plate protocol lists; the other gives the range a manufacturer describes as typical. If a protocol for your plasmid or strain names a specific concentration, follow that protocol, and for any new system check the range stated by the supplier of your own reagent.
Ampicillin versus carbenicillin
Addgene’s plate protocol states that carbenicillin can be used in place of ampicillin. It describes carbenicillin as more stable, possibly selecting better with fewer satellite colonies, and more expensive. The Addgene table lists carbenicillin at a 100 mg/mL stock and a 100 µg/mL working concentration, the same figures as ampicillin. See plasmid vectors for how selection markers fit into vector design.
Common misunderstandings
- “Add it before autoclaving.” The manufacturer page says not to autoclave ampicillin in media. Add it to sterile, cooled medium.
- “Add it to hot agar.” The Addgene protocol cools the molten agar for at least 5 minutes in a 60 °C water bath before the antibiotic goes in.
- “There is one universal number.” Sources state 100 µg/mL for plates and 50-100 µg/mL as the typical range in media, so cite the protocol you actually follow.
- “mg/mL and µg/mL are interchangeable.” They differ by a factor of 1,000; mixing them up gives a thousandfold error.
- “This is a patient dose.” It is not. A working concentration describes bacterial culture medium and says nothing about clinical use.
FAQ
What is the ampicillin concentration in LB broth?
100 µg/mL is the figure in Addgene’s LB plate protocol, and 50-100 µg/mL is the range one manufacturer calls typical for culture media.
How much ampicillin do I add to LB broth?
With a 100 mg/mL stock, add 100 µL per 100 mL of LB for 100 µg/mL, or 50 µL per 100 mL for 50 µg/mL.
Can I autoclave ampicillin with LB?
No. The manufacturer page says not to autoclave it in media; add it after the medium is sterile and cooled.
Can carbenicillin replace ampicillin?
Addgene’s protocol says it can, noting that it is more stable and more expensive.
Sources
- Addgene, Pouring LB Agar Plates, checked 2026-10-29.
- Teknova, Ampicillin 100 mg/mL (A2135) product page, manufacturer statement, checked 2026-10-29.
