Preparation of stock solution of tetracycline

Overview

  • 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.

Requirement

  • 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

Composition

  • 12 mg/ml tetracycline
  • 70% ethanol

Objective

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

Preparation

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.

Tips:

  • 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.

Precautions:

  • 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.

Storage

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

Precautions:

  • Protect the solution from light.

Applications

  • 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