Southern Blotting Lab Report

Edwin Southern invented the molecular technique of Southern Blotting in 1975. This technique is very useful because of its ability to detect a specific DNA sequence from a large amount of DNA ⎯ even from the whole genome. This specific sequence can be found through a combination of two different techniques: Agarose Gel Electrophoresis and Hybridization, which is known as Southern Blotting. This technique if performed in three phases: (I) prepare the gel and (II) make the blot, (III) hybridize and visualize blot.

In phase one, the gel is prepared in three steps: (1) chemical digestion, (2) electrical separation, and (3) chemical denaturation. In the first step, the DNA sample is digested with three restriction enzymes that were picked as part of a triple digest plan. The result of which can be used to make a restriction map of the DNA of interest. Secondly, after the digestion of the sample, the agarose gel can now be run to separate the fragments by size. In step three, which is performed twice, the gel from the previous step it soaked in a bath of sodium hydroxide (NaOH) and placed on a shaker. This denatures the DNA and makes it single stranded (ssDNA). Be sure, after the bath, that the base is neutralized before continuing on.
First, a solution of SSC is placed in a container, and paper towels are placed a tray with the ends of the paper in the solution to form a wick. Then a sandwich of the gel, a sheet of nitrocellulose, paper towels, and a weight (in that order from bottom to top) is placed on the tray. The transfer of the ssDNA fragments to the nitrocellulose membrane is achieved through capillary action, meaning that the solution moves from the container through the gel to the paper towels because of the cohesive property of liquids. The sheet of nitrocellulose with the copy of ssDNA fragments is now known as the