Preparation of Stock Solution of Tetracycline (12 mg/ml)

Overview

  • Tetracyclines are a group of broad-spectrum antibiotics. They are active against a wide range of microorganisms including bacteria (both gram-positive and gram-negative), chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites.
  • Tetracyclines function by inhibiting protein synthesis.
  • Binding of tetracyclines to 30S ribosomal subunit prevents 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. The stock solution is diluted to the 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 a 15 ml screw-capped tube. Add 9 ml of 70% ethanol. Mix until all tetracycline dissolves completely.

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

Precaution:
Do not dissolve in 10 ml of deionized / Milli-Q water. It is always a good practice to dissolve all ingredients first and then adjust the volume to the desired volume with the 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 a long time.

Precaution:
Protect the solution from light.

Applications
> A selection marker for bacterial culture during gene cloning experiments.
> Often used for inducible expression systems (tet-on tet-off) in mammalian cells.

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