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Gel electrophoresis procedure

In gel electrophoresis, a positive electrode is positioned at one end of the gel layer, and a negative cathode at the other. The gel layer is formed with small wells in one end, where the DNA fragments and reagent mixture is loaded (Step 1, Figure 1).

DNA is negatively charged. When a current is passed through the gel, the DNA moves through the pores in the gel towards the positive electrode (Step 2, Figure 1). Smaller DNA molecules move faster through the gel than larger DNA molecules, leading to size separation. This difference in the rate of migration separates the fragments on the basis of size (Step 3, Figure 1).

After electrophoresis, DNA is visualized and appears as 'bands' of grouped DNA fragments of the same length. Only a large amount of DNA (millions of copies) can be visualized with this method (Step 4, Figure 1).

The sizes of the DNA samples can be estimated by comparing the distance with the DNA ladder ('marker DNA' in Fig.1).

Labelled diagram showing the steps in gel electrophoresis. The plastic gel tray contains agarose gel. Several wells at the top of the gel allow for the insertion of the DNA samples. An electrical power supply of 80 to 120 volts is attached to the tray. In the first step, the DNA samples, in gel loading buffer, are loaded into the marked wells on the tray using a pipette. In the next step, the power source is activated to apply an electrical field. The DNA samples have a negative charge, and a positive charge is created at the opposite end of the tray from where the sample is loaded. In the next step, DNA separation occurs. Over 30 to 45 minutes, the samples are left to separate according size. A DNA marker, or ladder, is loaded into one field of the tray. The marker contains fragments of differing sizes and can be used to estimate the size of the fragments in the DNA samples. During separation, the DNA fragments in each sample travel down the tray towards the anode, stopping at different points depending on their size. In the last step, UV transillumination and documentation means that the fragments can be visualized as bands in the gel tray.

Figure 1. Fragments of different lengths from a PCR reaction are run on a gel. The fragments will move at a speed and a distance relative to their size: smaller DNA molecules will move in the gel faster and further than longer DNA molecules.

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Gel electrophoresis preparation