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Standard DNA fragments Sample (i.e., kilobase wells ladder)

Standard DNA fragments Sample (i.e., kilobase wells ladder)

FIGURE 21.05 Agarose Gel Separation of DNA: Staining and Standards

To visualize DNA, the agarose gel containing the separated DNA fragments is soaked in a solution of ethidium bromide, which intercalates between the base pairs of the DNA. Excess ethidium bromide is removed by rinsing in water, and the gel is placed under a UV light source. The UV light excites the ethidium bromide and causes it to fluoresce orange. In sample A, there are two fragments of DNA each of a different size and each forming a separate band in the gel. To determine the size of fragments, a standard set of DNA fragments of known sizes is run alongside the sample to be analyzed.

intercalates between the base pairs of DNA and RNA, therefore, DNA binds much more molecules of ethidium bromide than RNA. The gel must then be examined under UV light, where ethidium bromide bound to DNA fluoresces bright orange. RNA also fluoresces under UV light, but not as intense. After the bands are located, they may be cut out of the agarose slab and the DNA extracted to yield a pure fragment.

Agarose gel electrophoresis can be used to purify DNA for use in genetic engineering or it can be used to measure the sizes of different fragments. To find the size of an unknown piece of DNA, a set of standard DNA fragments of known sizes is run alongside, on the same gel (Fig. 21.05). A set of DNA fragments that are exact multiples of 1000 bp are often used. This is known as a kilobase ladder.

Very large DNA molecules may be separated by pulsed field electrophoresis.

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