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Southern Blotting Techniques - The original method was first developed by E. M. Southern in 1975 and later was modified by various people for various applications. The process of Southern blotting starts after the agarose gel electrophoresis of restriction digest or gDNA. The gel is first washed with buffer to remove the broken or fragmented residues of agarose formed during banding and the accumulated contaminants.

Then the gel is placed in acid solution (0.25 M HCI) for about 5-10 min. During this step, the DNA undergoes depurination. After this step the gel is placed in 0.25 M NaOH alkali solution. This step ensures that the DNA is shorter in length (<500 bp) and is in single-stranded form. DNA fragments large in size (>10 kb) require a longer time and move in zig-zag form when compared to shorter DNA fragments.

Finally, the gel is extensively washed with buffer to remove the traces of HCI and NaOH. Then the gel is placed on the filter paper with wigs dipped in a reservoir containing transfer buffer placed in transfer buffer reservoir tank which is placed on the glass plate.

Nitrocellulose or nylon filter paper is placed on the gel above which a stack of blotting papers soaked in transfer buffer is placed and gently pressed using glass plate or rod to remove the air that is trapped between the gel and membrane. Then a stack of unsoaked blotting papers is placed above them and the glass plate with a weight of 500 g is placed above it.

By capillary action the transfer buffer is transferred from the reservoir to the blotting papers. During this process the DNA from the gel is transferred onto the nitrocellulose paper and immobilized there due to the negative charge of the DNA and the positive charge of the membrane.

This set up is allowed to stand like that for 12-18 hours. After this time period the nitrocellulose or nylon membrane is taken out from the set up carefully. As the binding of the DNA to the nitrocellulose or nylon membrane is weak, it has to be fixed more strongly, so that the DNA is not washed off during the washing of the membranes. There are two widely used methods for this procedure.
After the transfer, the membrane is exposed to ultraviolet rays, so that a bond is formed between thymine residues present in the DNA and the positively charged amino groups on the surface of the nylon membrane. This method is called as UV­cross linking method. The other method is baking of the nitrocellulose or nylon membrane at 80°C in a vacuum oven.

The use of vacuum oven is a must due to the flammable nature of nitrocellulose membrane. At present, various advances have been made in the technique of Southern blotting, the first and foremost being the use of electricity instead of capillary action as a force for transferring the DNA from the gel onto the membrane.
The second and most significant is the use of positively charged nylon membrane, which reduces the need for excessive depurination. These two alternative methods have significantly reduced the time and damage to DNA during the process.
 

 

 
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