Gene Cloning

Key Tools for Gene Cloning 61
Figure 3.17 Photograph of agarose gel. Lane M shows marker DNA in this case
the 6 largest bands produced by cutting bacteriophage λ DNA with HindIII. The
other lanes show DNA fragments ranging in size from 2 to 7 kb.
monitoring the progress of electrophoresis, and a dense liquid such as
glycerol, which ensures that the sample settles into the bottom of the well.
The DNA samples are loaded into the wells and a current applied. The DNA
molecules thread their way through the gel and are separated according to
size. The DNA is visualized by staining with ethidium bromide, which fluoresces
under ultraviolet light. This dye intercalates between the bases of
DNA, which concentrates it in the gel where the DNA is. As a result these
bands fluoresce brightly under ultraviolet illumination. This staining technique
is very sensitive; as little as 0.05 μg of DNA in one band can be
detected. Despite the fact that ethidium bromide is a mutagen and ultraviolet
light can cause severe burns this is still the most commonly used
technique for visualization of DNA.
The distance that the band has migrated from the well is a measure of
the size of the DNA fragments. Gels are calibrated by running a sample of
DNA, which contains a series of restriction fragments of known size, in parallel
to the experimental samples. A commonly used DNA size marker is
DNA from the bacteriophage lambda, cut with HindIII, which gives a recognizable
pattern of eight bands ranging in size from 125 bp to 23.13 kb
(Figure 3.17). The relationship between the size of the DNA fragments and
the distance the bands migrate is not a linear one; rather, the distance