Agarose Gel Electrophoresis: Principles, Structure, Procedure, and Applications

I. Introduction

When an electric current is applied to a medium containing charged molecules, these molecules migrate toward the electrode with opposite polarity. Depending on the medium and the physical properties of the analytes (e.g., size and shape), this migration results in the spatial separation of molecules. This is the foundational principle behind electrophoretic techniques such as agarose gel electrophoresis (AGE).

II. What Is Agarose Gel Electrophoresis?

Agarose gel electrophoresis separates nucleic acid fragments based on their size. Negatively charged DNA/RNA molecules migrate through the gel matrix toward the positive electrode (anode). Smaller fragments migrate more rapidly than larger ones.

III. Principle of Agarose Gel Electrophoresis

Agarose is a purified linear polysaccharide. When cooled from a sol state, it forms a thermoreversible 3D network via hydrogen bonding. This porous structure acts as a molecular sieve.

Pore Size and Concentration

Pore size is inversely proportional to agarose concentration. Below is a selection guide for gel percentages:

Gel Matrix Formation

DNA Migration Principle

IV. Procedure

1. Gel Preparation

Dissolve agarose in TAE/TBE buffer. After heating, add Ethidium Bromide (EtBr) or safer alternatives once cooled to ~55°C.

2. Loading & Electrophoresis

Run the gel at 1–5 V/cm. Ensure the wells are at the cathode (-) side.

3. Visualization

V. Key Applications