Southern Blot

Southern Blot analysis of DNA

    The left panel is an electrophoretic gel stained with ethidium bromide. Total DNA has been extracted from 9 plasmid clones (lanes ##1-9), digested with a particular restriction endonuclease, and separated according to size by electrophoresis. The DNA appears smeared, with no discrete bands, as no one DNA sequence is present in more than one copy. The molecular weight standard (a "Ladder") has a series of DNA fragments of known size. A cloned DNA fragment has been loaded in lane #10: the weight standard indicates a size of about 400bp.

    The middle panel is a Southern Blot autoradiogram of the same gel. The DNA in the gel has been transferred ("blotted") to a filter, then the filter is exposed to ("probed") 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 homologous sequence in the blot, it base-pairs ("hybridizes") 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 interpretation of the Southern Blot autoradiogram: the information content is the presence or absence of bands, and their sizes. The presence of a band shows that a DNA sequence homologous to the probe DNA is present in clones ## 3, 4, & 8, with the same size as the probe, and absent in clones ##1 ,2, 5, & 9.  The probe sticks to itself in #10, as expected. In lane #6, a homologous sequence is present, but with an additional restriction site that cuts the fragment into two smaller fragments of 300bp and 100bp. This analysis indicates that the gene of interest has been successfully cloned in a particular set of plasmids, and that there is genetic variation in clone #6. This gene can now be analyzed further.

Figures modified from © 2000 by Griffiths et al.; text material © 2008 by Steven M. Carr