Southern Blot

Uniformed Services University

Department of Microbiology and Immunology

Used by the Laboratory of William C. Gause, Ph.D..,

Southern Blotting The first blotting technique to be devised is known as Southern blotting after its originator E. M. Southern.

This technique is capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavage of the entire human genome with a restriction enzyme. In such a complex mixture, many fragments will have the same or nearly the same length and thus migrate together during electrophoresis. Even though all the fragments are not separated completely by gel electrophoresis, an individual fragment within one of the bands can be identified by hybridization to a specific DNA probe. To accomplish this, the restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting.

This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter, much like the replica filter produced from clones in a library. (The blot is used because probes do not readily diffuse into the original gel.) The filter then is incubated under hybridization conditions with a specific radiolabeled DNA probe, which usually is generated from a cloned restriction fragment. The DNA restriction fragment that is complementary to the probe hybridizes, and its location on the filter can be revealed by autoradiography.

Southern Blots

This method is used pro the identification of genetic material fragments with the intention of are separated on an 1% agarose electrophoresis gel. Inside opinion it is akin to the western mark single with the intention of we are using labeled DNA to detect the denatured DNA transferred from the gel to the blotted crust.  The crucial step is to take an “image” of the gel previous to transferring the DNA in the gel to a nylon crust. This conveying (or blotting) is held to keep up the relation location of all genetic material fragments and this will single bring about with denaturing the DNA fragments with sodium hydroxide (breaking up dual strands). This could require the attention of pressure or other methods to proceed to completion. Inside addition, small concentrations on the gels must trade show on the mark.

The single stranded DNA fragments on the crust are at that time immobilized by excitement or UV light exposure. Detection of point sequences occurs by hybridization. A sequence of DNA or RNA, which carries a detectable label, complementary to the DNA fragment of appeal is useful to the crust. Chromogenic, fluorogenic or radiolabels can be used.

After washing and the attention of the corresponding stain reagent, hybridized bands on the crust are visible and they can at that time be associated with the bands on the first gel.

Southern blot protocol

Required Solutions

RUNNING THE GEL

GEL TRANSFER

PREHYBRIDIZATION/HYBRIDIZATION

Other Protocol

Southern blot protocol