7.4. Agarose gel electrophoresis

As discussed in the introduction, the pore size of the gel defines the operational molecular mass range in which effective separation can be achieved. The size of the DNA molecules most frequently handled in a typical recombinant DNA experiment is in the range of thousands of base pairs (see in Chapter 10). Such very large molecules cannot be separated in polyacrylamide gels, as even the largest pore sizes achievable in such gels are too small for these macromolecules. Naturally, a different type of gel matrix is needed.

For this purpose, agarose has become the most popular matrix. The agarose gel forms through non-covalent interactions between polysaccharide molecules. When such a gel is heated, it undergoes a phase transition from gel to sol state. The agarose powder is mixed with running buffer and the slurry is heated up to reach the sol state. The liquid is then poured in an appropriate, usually horizontally-mounted, template to solidify.

The pore size is set by the concentration of agarose monomers. The agarose gel meets all of the important requirements that make a good electrophoresis matrix: it is hydrophilic, does not carry charges, chemically inert and does not absorb the dyes used for nucleic acid staining.