The probe will bind with the desired DNA molecule by making it ds-DNA. Then, hybridize the DNA bands of the nylon filter by adding radioactive probe in situ. Sometimes, UV-treatment is also employed to facilitate the cross-linking of the DNA strands to the filter membrane. UV-treatment or bakingĪfter blotting, bake the nitrocellulose filter membrane at 80 degrees Celsius for up to 10 minutes. The exact bands on agarose gel will now appear on the filter paper through the capillary action. The transfer of DNA bands to the nitrocellulose filter is the process of forming replicas. Then, place the nitrocellulose filter membrane and add some paper towels or some weight over the top of the filter membrane.After that, place the agarose gel matrix consisting of different DNA bands.First, take the alkaline buffer solution in the container.Blotting is a very crucial step which has to be performed with care and it involves the following steps: Southern blotting involves the transfer of the ss-DNA bands from the agarose gel to the nitrocellulose filter paper. Depurination makes the use of acidic solution (0.2 M HCl). To reduce the size or the length of the separated strands, a technique called depurination is employed. For denaturation, 0.5 M NaOH solution is generally used. DenaturationĪfter the band formation, subject the gel matrix to the alkaline solution to denature the DNA or to break the ds-DNA into ss-DNA. Therefore, different bands of DNA will appear of varying length on the gel matrix. The larger DNA fragments will found close to the well, whereas the smaller DNA fragments will move faster. The gel matrix provides the complex network for the migration of DNA fragments based on their size from a cathode to anode under the influence of an electric field. Then, separate the DNA of different size or length on the agarose gel medium by gel electrophoresis. Finally, the extracted DNA molecule is cleaved into short fragments by the enzyme restriction endonuclease. Then, the extracted DNA is purified by alcohol precipitation. It results in the expulsion of the DNA from the ruptured cell’s nucleus. Before restriction digestion, homogenization of the sample is performed to facilitate entire cell lysis by using detergent. Southern blotting mainly involves the following seven steps: The filter membrane makes the further steps easier to perform like probe hybridization and autoradiography. The gel is fragile, whereas the nylon filter is easy to handle and acts as a sticky membrane where the DNA bands stick easily. The paper towel and weight do not allow the migration of DNA from the nylon filter. The capillary action moves the buffer solution upwards to the nitrocellulose or nylon filter, which will hit the DNA to be print on the filter. The replicas of DNA on the agarose gel is transferred to the nylon filter by the capillary mechanism. Transferring Mechanism of Southern Hybridization The probe binds with the target DNA can be visualized after exposing it to X-ray film by autoradiography. The probe is short, ss-DNA and labelled with a radioactive isotope. The target DNA reacts with a specific DNA probe, which results in a complementary pairing of ss-DNA with the radioactive probe. The southern hybridization is carried out to separate desired DNA. Southern blotting is done after the separation of DNA fragments based on length by electrophoresis. Southern hybridization is based upon separating the target DNA through probe hybridization and autoradiography. This method is very much similar to the restriction fragment length polymorphism ( RFLP). Southern hybridization is a method of isolating DNA of interest from the mixture of DNA molecules. The probe complementary binds with the target DNA that can be visualized on the X-ray film. Thus, it isolates target DNA or desired gene of a sequence by labelling it with the specific radioactive probe. It can define as the immobilization of the DNA fragments from the agarose gel matrix to the solid support matrix (nitrocellulose filter or nylon) to detect specific genes or sequences by using radioactive probes. Southern hybridization is also called Southern blotting. Transferring Mechanism of Southern Hybridization.You will get to know the definition, principle, transferring mechanism, process and applications of the southern blotting method. The target ss-DNA then complementary pairs with the radioactively labelled DNA probe to form a ds-DNA, which can be visualized by the X-ray film. The nitrocellulose filter membrane containing the desired DNA sequences is further subjected to UV- treatment or baking to facilitate cross-linking of the DNA to the membrane. The target DNA is then immobilized from the agarose gel to the solid support matrix (nitrocellulose filter membrane) via capillary or electroblotting.

Northern hybridization