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 in each lane, 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 in a haploid bacterium. The lane marked (L) is a "Ladder", a molecular weight standard with a series of DNA fragments at 100 bp intervals. A cloned DNA fragment has been loaded in lane #10: its mobility corresponds to the fourth rung of the ladder, about 400 bp.

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

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, in each lane. The autoradiogram show that a DNA sequence homologous to the probe DNA is present in clones ## 3, 4, & 8, with the expected size as indicated in lane #10. The DNA is absent in clones ##1, 2, 5, & 9. The probe sticks to itself in control lane #10, as expected. The analysis shows that the gene of interest has been successfully cloned in the first set, which can now be analyzed further.