ANDC DU/Biology Protocols/Southern blotting

To perform southern blotting of DNA fragments from Agarose gel.

Introduction:

A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the British biologist Edwin Southern.

Principle:

It is a technique for transferring DNA molecules from agarose gel to a solid support such as nitrocellulose filter or nylon membrane. Gel embedded DNA is depurinated in the presence of HCl followed by denaturation in alkali. Denaturation prior to blotting is essential for dissociating the two polynucleotide chain which are then hybridized with the probe. Denatured molecules move upward by capillary action of buffer and on coming in contact with the nylon membrane they get bound to it. A brief UV treatment facilitates firm binding of DNA with the membrane.

Materials and Reagents :

Agarose gel containig DNA fragments Nylon membrane Whatman 3 mm paper Glass tray Rough Filter papers 0.25 M HCl Denaturing Solution Neutralizing Solution 10x SSC

Procedure:

 Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.  The DNA fragments are then electrophoresed on an agarose gel to separate them by size.  The gel was treated with HCl (0.25 M) for 15 minutes and then rinse with water, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.  DNA is denatured by adding sufficient volume of denaturing solution. Stir for 30 min.  Alkali was decanted and gel was rinsed with distilled water.  The gel was then neutralized in neutralizing buffer for 30 min.  Washed the gel with 2x SSC  A bridge was made on the glass tray with the glass plate and placed two layers of whatman 3 mm paper cut to proper size on top so that the ends of the paper hang down nto the tray. Wet the paper with 10x ssc and fill the tray with the same solution.  Another whatman paper is placed on the bridge which s cut to proper size of the gel and prewetted with 10xSSC.  The gel was then transferred on the top of the filter paper avoiding the bubbles.  Nylon membrane is then placed on the gel. The membrane is maked on the left hand bottom corner with a permanent ink marker. Wet the membrane in 2xssc and placed on the top of the gel.  The membrane is covered with two pieces of whatman paper presoaked in 2xssc  A stack of dry rough paper is placed on the top of wet filter paper.  A glass plate is placed on top of stack f filter papers and then put a weight of about 0.5 Kg and transfer was allowed to take place O/N.  Membrane was carefully removed and washed in 2xssc  Membrane was exposed to UV for not more than 2 min.  A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane.  The membrane is then baked, i.e., exposed to high temperature (60 to 100 °C) (in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to permanently and covalently crosslink the DNA to the membrane.

Precautions:

Use gloves while handling the gel and membrane Take care that no air bubbles are entraped between gel, membrane and whatman paper

Work in progress, expect frequent changes. Help and feedback is welcome. See discussion page.