Electrophoresis requires the use of a buffer system. A buffer is a chemical system that maintains a relatively constant pH even when strong acids or bases are added. Buffer solutions contain either a weak acid or a weak base and one of their salts. Buffers not only establish a pH, but provide ions to support conductivity. During electrophoresis, the electric field electrolyses the water molecules into H⁺ and OH^- ions that migrate to the respective migrate to the respective electrodes.

The increase in H⁺ and OH^- alters the pH at each electrode. A buffer can effectively neutralize the ions so that the pH of the system is maintained. The makeup of the buffer system is critical in the separation of the amplified DNA products by ensuring that the sample molecules are ionized and controlled. Changes to the buffer system can lead to poor separation and lack of reproducibility. Small changes in pH can change molecular charge; whereas, large changes in pH can cause serious, irreversible changes in molecular structure. Nucleic acids are relatively unaffected by pH changes due to their negative charge.⁰⁴

The choice of buffer system will affect the resolution of the components in the mixture. Both continuous and discontinuous buffer systems can be used.

A continuous system uses the same buffer for the tank and gel. In continuous systems, molecular charge and gel pore size are the only factors that have any effect on the separation and stacking or concentration of a sample into a band. A concentrated band of sample forms where the molecules are slowed down at the interface of the buffer and the gel. Since smaller molecules have less of a difference between their free solution mobility and their mobility in a gel, the stacking of smaller fragments is not as favorable as it will be in a discontinuous buffer system.

In a discontinuous system, the tank and gel buffers are different from each other. Samples are loaded onto a large-pore gel, called a stacking gel, which overlays a smaller pore resolving gel. The stacking gel serves to concentrate the DNA molecules on top of the resolving gel. After entering the resolving gel, the DNA molecules are separated according to molecular size. The major advantage of a discontinuous buffer system is the increased resolution and concentration of the sample band.

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