southern blotting technique

The name of this technique is derived from the following:

• The name of its inventor, E.M.Southern
• The DNA-DNA hybridization that forms its basis.

It is also called “Southern blotting”, since the procedure for transfer of DNA from the gel to the nitrocellulose filter resembles blotting.

DNA sample is first digested with a restriction enzyme and digested sample is gel electrophoresed. The DNA bands in the gel are denatures into single strands with the help of an alkali solution.subsequently, the gel is laid on top of a buffer saturated filter paper, placed on a solid support, with its two edges of the filter paper immersed in the buffer. A sheet of nitrocellulose membrane is placed on top of the gel and a stack of many papers on the top of this membrane. 500 grams weight placed on the top of paper towels.

The buffer solution moves, due to capillary action, from the bottom filter paper through the gel carrying with it the denatured DNA present in the gel; the DNA becomes trapped in the nitrocellulose membrane as the buffer phases through it. This process is known as “blotting” and takes several hours to complete.

While passing through the gel, the buffer carries with it single stranded DNA, which binds on to the nitrocellulose membrane, when the buffer passes through it to the paper towels. After leaving this arrangement for a few hours (or) overnight, paper towels are removed and discarded. The nitrocellulose membrane with single stranded DNA bands blotted on to it., is baked at 80⁰C for two to three hours to fix the DNA permanently on the membrane. This membrane now has a replica of DNA bands from agarose gel, and can be used for hybridization with radioactively labelled DNA (or) RNA probe. The membrane may then be washed to remove any unbound DNA and X-ray film is exposed to the hybridized membrane to get autoradiograph.

 

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Southern Blotting Video