Separating DNA Fragments: Gel Electrophoresis

Separating DNA Fragments: Gel Electrophoresis

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As we saw in the previous item, DNA fragments formed with the action of restriction enzymes have different sizes. The most widely used technique for separating DNA fragments is electrophoresis through agarose gels.

Agarose is a polysaccharide (such as agar and pectin) that dissolves in boiling water and then gels when it cools down like gelatin. To perform an electrophoresis, an agarose gel is prepared, DNA is introduced into small gel wells, and then an electric current is applied through the gel. As the DNA is negatively charged, it is attracted by the positive electrode. However, to reach the positive electrode, DNA must migrate through the agarose gel.

Smaller DNA fragments can migrate through an agarose gel faster than larger DNA fragments. The migration rate of linear DNA fragments through agarose is inversely proportional to log10 of their molecular weights.

You can calculate the exact size of a given fragment based on its migration ratio. After gel electrophoresis, DNA fragments are usually stained with ethidium bromide, which has affinity for DNA and fluoresces (becomes visible) vividly in contact with ultraviolet light. This way you can locate the bands that correspond to the DNA. DNA fragments can then be isolated and purified from agarose gels.

And now? What do you do with these DNA fragments?