Antibiotic Resistance Genes and Selection Concentrations
Most cloning vectors carry an antibiotic resistance gene as a selection marker, so only cells that took up the plasmid grow on selective medium. The tables below list the common markers, their mechanism and the working and stock concentrations used in the lab. These are commonly used ranges for E. coli (and standard mammalian selection) — optimise per strain, plasmid copy number and protocol.
Bacterial (E. coli) selection
| Antibiotic | Resistance gene (marker) | Mechanism | Working conc | Stock | Solvent | Notes |
|---|---|---|---|---|---|---|
| Ampicillin | bla (AmpR) | β-lactamase (degrades β-lactam) | 100 µg/mL | 100 mg/mL | water | Secreted enzyme → satellite colonies; light/heat labile |
| Carbenicillin | bla (AmpR) | β-lactamase (more stable analog) | 50–100 µg/mL | 50 mg/mL | water | Fewer satellite colonies than ampicillin |
| Kanamycin | aph(3′) / neo (KanR) | aminoglycoside phosphotransferase | 50 µg/mL | 50 mg/mL | water | — |
| Chloramphenicol | cat (CmR) | acetyltransferase | 25–34 µg/mL | 25–34 mg/mL | ethanol | Bacteriostatic |
| Tetracycline | tet (TetR) | efflux pump | 10–15 µg/mL | 5–10 mg/mL | ethanol (or 70% EtOH) | Light-sensitive; chelates divalent cations |
| Spectinomycin | aadA (SpecR) | adenylyltransferase | 50–100 µg/mL | 50 mg/mL | water | — |
| Streptomycin | aadA / strA-strB | aminoglycoside modification | 25–50 µg/mL | 50 mg/mL | water | — |
| Gentamicin | aacC1 (GmR) | aminoglycoside acetyltransferase | 10–20 µg/mL | 10 mg/mL | water | — |
| Zeocin | Sh ble (ZeoR) | binds/cleaves DNA (drug sequestration) | 25–50 µg/mL | 100 mg/mL | water | Use low-salt LB ~pH 7.5; light-sensitive |
Mammalian cell selection
| Drug | Resistance gene | Typical working conc | Notes |
|---|---|---|---|
| G418 / Geneticin | neo / aph | 100–800 µg/mL | Aminoglycoside; titrate with a kill curve |
| Hygromycin B | hph | 50–200 µg/mL | — |
| Puromycin | pac | 1–10 µg/mL | Fast selection (days) |
| Blasticidin S | bsr / bsd | 2–10 µg/mL | — |
| Zeocin | Sh ble | 100–400 µg/mL | Also works in bacteria/yeast |
These ranges are commonly cited starting points — verify for your strain, vector copy number and protocol.
How antibiotic selection works
The plasmid carries a resistance gene whose product inactivates, modifies, pumps out or otherwise neutralises the antibiotic. When you plate transformed cells on medium containing the drug, only cells that took up the plasmid — and therefore express the marker — survive and form colonies. Cells without the plasmid are killed or held back, so selection enriches for your construct.
Stocks and handling
Stocks are typically made at 1000× the working concentration (e.g. 100 mg/mL ampicillin for a 100 µg/mL plate), filter-sterilised and stored at −20 °C. Add antibiotics to autoclaved media only after it has cooled below ~55 °C — pouring into hot agar destroys heat-labile drugs. Several are light- or heat-sensitive and should be protected from light and used reasonably fresh, notably ampicillin, tetracycline and Zeocin.
Bacterial vs mammalian selection
Bacterial and mammalian systems use different drugs and markers, and mammalian selection runs at much higher concentrations — hundreds of µg/mL for agents like G418 versus tens of µg/mL in E. coli. Because the lethal dose varies widely between cell lines, you should always run a kill curve for mammalian selection: test a range of concentrations on untransfected cells and choose the lowest dose that kills them within the expected window.
Frequently asked questions
- What is a selection marker?
- A selection marker is a gene carried on a plasmid (or integrated construct) that lets cells survive a drug they otherwise could not. On selective medium, only cells that took up the plasmid grow, so the marker selects for successfully transformed or transfected cells. The most common bacterial markers are bla (ampicillin), neo/aph (kanamycin) and cat (chloramphenicol).
- What is the working concentration of ampicillin?
- For E. coli, ampicillin is commonly used at 100 µg/mL, typically prepared from a 100 mg/mL stock in water (a 1000× stock). This is a commonly cited starting point — verify for your strain, vector copy number and protocol.
- Why do I get satellite colonies?
- Ampicillin is degraded by the secreted β-lactamase enzyme that resistant colonies produce. As the drug breaks down around a growing colony, nearby non-resistant cells survive and form small 'satellite' colonies. Using carbenicillin (a more stable analog) or picking colonies before plates over-grow reduces satellites.
- What solvent do I use for chloramphenicol and tetracycline?
- Chloramphenicol and tetracycline stocks are dissolved in ethanol (tetracycline in ethanol or 70% ethanol), not water. Tetracycline is light-sensitive and chelates divalent cations, so protect stocks from light. Most aminoglycosides (kanamycin, gentamicin) and β-lactams dissolve in water instead.
- Why are mammalian selection concentrations so much higher?
- Mammalian cells tolerate far higher drug levels than E. coli, so selection agents such as G418 are used at hundreds of µg/mL rather than tens. The exact lethal dose varies by cell line, so you should always run a kill curve — testing a range of concentrations on untransfected cells — to find the lowest dose that kills them.
See also
Related tools and references
Use these related pages when this table raises a practical calculation or workflow question.