Southern Blot analysis

Southern Blot analysis of DNA

The left panel is an electrophoretic gel stained with ethidium bromide. Total DNA has been extracted from nine bacterial DNA clones (lanes ##1-9), digested with a particular restriction endonuclease, and separated by electrophoresis. Starting about one-third of the way from the top, a bright smear is present, indicating the presence of a random collection of restriction fragments of various sizes. There are no discrete bands, as no one DNA sequence is present in more than one copy. The lane marked (L) is a molecular weight standard (a "Ladder"), with a series of DNA fragments at 100bp intervals. A cloned DNA fragment has been loaded in lane #10: its mobility corresponding to the four rung of the ladder indicates a size of about 400bp.

The middle panel is a Southern Blot autoradiogram of the same gel. The DNA in the gel is transferred ("blotted") to a filter. The filter is then hybridized with a radioactively-labelled DNA (a "probe") made from the DNA loaded in lane #10. The filter is then exposed to X-ray film. Where the probe DNA finds a complementary sequence in the blot, it base-pairs ("sticks") to that DNA. The radioactivity then exposes the film and produces a dark band on the X-ray film.

The right panel is a schematic representation of the autoradiogram: the information content is the presence or absence of bands, and the size of the fragments. The bands in the autoradiogram show that a DNA sequence homologous to the probe DNA is present in clones ## 3, 4, & 8, with the expected size, and absent in clones ##1, 2, 5, & 9. [The probe sticks to itself in #10, as expected]. This analysis indicates that the gene of interest has been successfully cloned in the first set of plasmids, which can now be analyzed further.