Antibiotic Resistance Genes & Concentrations

Mammalian cell selection

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 neutralizes 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-sterilized 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.

See also

• Restriction Enzymes — recognition sites and cut positions →
• Restriction Enzyme Digest Guide →

Sources

1. 1
   Antibiotics (molecular biology reference table)

   Addgene

   The Working conc / Stock / Solvent columns of the bacterial (E. coli) table, for the rows it carries: ampicillin 100 mg/mL stock and 100 ug/mL working; carbenicillin 100 ug/mL working; kanamycin 50 mg/mL and 50 ug/mL; chloramphenicol 25 mg/mL in ethanol and 25 ug/mL; tetracycline 10 mg/mL and 10 ug/mL; spectinomycin 50 mg/mL and 50 ug/mL. It also backs the FAQ answers 'ampicillin is commonly used at 100 ug/mL from a 100 mg/mL stock (a 1000x stock)' and the ethanol solvent for chloramphenicol and tetracycline. If a second, more formally citable reference is wanted for the stock solutions, Cold Spring Harbor Protocols publishes the Molecular Cloning recipes with DOIs — e.g. 'Ampicillin stock solution (100 mg/mL)', doi:10.1101/pdb.rec11179, which I fetched and which specifies 1 g sodium ampicillin in 10 mL H2O, 0.22 um filtered, stored at -20C.
2. 2
   Selection Antibiotics for mammalian cell culture

   Thermo Fisher Scientific (Gibco)

   The mammalian cell selection table and the 'always run a kill curve' advice in both the FAQ and the 'Bacterial vs mammalian selection' section. Thermo's stated ranges: Geneticin/G418 200-500 ug/mL, Hygromycin B 100-1,000 ug/mL (typically 200), puromycin 0.2-5 ug/mL, blasticidin 1-20 ug/mL, Zeocin 50-400 ug/mL, with explicit instruction that the concentration be optimised per cell line by dose-response. Read the discrepancy note in `unsourced` before citing this as backing the exact numbers shown.
3. 3
   Molecular mechanisms of antibiotic resistance

   Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ · Nature Reviews Microbiology 13(1):42-51 · 2015

   The Mechanism column of the bacterial table as a class: drug-inactivating hydrolysis (bla / beta-lactamase, for ampicillin and carbenicillin), aminoglycoside-modifying enzymes by phosphorylation, acetylation and adenylylation (aph(3')/neo for kanamycin, aacC1 for gentamicin, aadA for spectinomycin/streptomycin), acetyltransferase inactivation (cat, chloramphenicol) and active efflux (tet). It also backs the 'How antibiotic selection works' section. This is a REVIEW, chosen deliberately: the alternative is eight separate original papers for one table column. If a more specific citation is wanted for the aminoglycoside rows, Ramirez MS & Tolmasky ME, 'Aminoglycoside modifying enzymes', Drug Resist Updat 13(6):151-171 (2010), doi:10.1016/j.drup.2010.08.003, names the APH(3'), AAC and ANT/aadA families directly.
4. 4
   Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering

   Dumas P, Bergdoll M, Cagnon C, Masson JM · EMBO Journal 13(11):2483-2492 · 1994

   One cell that the general mechanism review does not reach: the Zeocin row's mechanism, 'binds/cleaves DNA (drug sequestration)' for Sh ble (ZeoR), in both the bacterial and mammalian tables. Sh ble confers resistance by binding the bleomycin-family drug stoichiometrically and sequestering it, rather than by modifying or expelling it — which is why this row's mechanism is phrased differently from every other row.

Related tools and references