Preparation of stock solution of tetracycline


  • Tetracyclines are a group of broad-spectrum antibiotics. They are active against wide range of microorganisms including bacteria (both gram positive and gram negative), chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites.
  • Tetracyclines function by inhibiting the protein synthesis.
  • Binding of tetracyclines to 30S ribosomal subunit prevent access of aminoacyl tRNA to the acceptor site on the mRNA – ribosome complex, leading to inhibition of protein synthesis.
  • Tetracycline hydrochloride can be used for biological research.
  • Tetracycline hydrochloride is soluble in water (50 mg/ml) with heating.
  • It forms a clear, yellow to yellow-orange solution in water.
  • It also forms a clear, yellow-green solution in 95% ethanol.
  • Tetracycline is rapidly destroyed by alkali hydroxide solutions and solutions in water become turbid on standing due to hydrolysis and precipitation of tetracycline.
  • The potency of tetracycline is reduced in solutions with pH below 2.
  • A 5 – 12 mg/ml stock solution of tetracycline is generally prepared in 70% ethanol. Stock solution is diluted to final concentration, depending on the need.
  • Magnesium ions are antagonistic to tetracycline, therefore, don’t use magnesium salt in bacterial culture medium if tetracycline is going to be used for selection. This may lead to false positives.


  • Reagents
    • Tetracycline hydrochloride (C2H24N2O8.HCl) (Molecular weight: 480.9)
    • 70% ethanol
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker / 15 ml screw-capped tube
    • Magnetic stirrer


  • 12 mg/ml tetracycline
  • 70% ethanol


  • Preparation of 10 ml of tetracycline stock solution (12 mg/ml) in 70% ethanol


Step 1: To prepare 10 ml of 12 mg/ml tetracycline stock solution, weigh out 120 mg of tetracycline. Transfer it to 15 ml screw-capped tube. Add 9 ml of 70% ethanol. Mix until all tetracycline dissolves completely.


  • We recommend to use 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 9 ml 70% ethanol and mix the content by rotating the tube. Once the content is mixed, make up the volume 10 ml.


  • Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.

Step 2: Adjust the volume to 10 ml with 70% ethanol. Mix it again.


Store the solution in small aliquots (1 ml) at -20°C for long time.


  • Protect the solution from light.


  • A selection marker for bacterial culture during gene cloning experiments.
  • Often used for inducible expression system (tet-on tet-off) in mammalian cells.
Follow the table to prepare tetracycline stock solution of various volume (5 ml, 10 ml, 25 ml, 100) and concentration.
Conc. / Volume 5 ml 10 ml 25 ml 100 ml
5 mg/ml 25 mg 50 mg 125 mg 500 mg
10 mg/ml 50 mg 100 mg 250 mg 1000 mg
12 mg/ml 60 mg 120 mg 300 mg

1200 mg