The membrane is then incubated with a labeled probe which contains the complementary sequences to the gene of interest on the membrane.The size of the DNA fragments can be determined by comparing their relative size with the DNA bands of known lengths.The position of the band containing these hybridized fragments is determined by autoradiography or any other method depending on the types of probes used.The probe hybridizes with the immobilized ssDNA on the membrane having the gene of interest.The membrane is then incubated with an appropriate radiolabeled probe (radiolabeled ss DNA or radiolabeled RNA) specific for the gene of interest.After transfer, the strands on the membrane surface are immobilized by baking or UV-radiation.The resulting single stranded DNA is then transferred onto a nitrocellulose or nylon membrane either by capillary blotting or by electroblotting.The gel is then placed in alkaline salt solution to denature the double stranded DNA.In Southern blotting, DNA is first cut with restriction enzymes and the fragments are separated according to size by agarose gel electrophoresis.This process of base pairing between complementary single-stranded DNA molecules is known as hybridization.This technique is based on the formation of hybrid DNA molecules when homologous, denatured DNAs from two different sources are mixed with each other under the appropriate conditions of ionic strength and temperature.You may also want to see: Western blotting: Principle, Steps involved and Applications This technique helps in the identification of a particular DNA fragment containing the gene of interest.This technique is named after its inventor Edward N.Southern blotting also called Southern hybridization is a hybridization technique which helps to detect specific gene sequences or DNA fragments within complicated mixture of nucleic acids.The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer. ![]() This slows down the blotting process and may reduce the amount of DNA that can be transferred. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. A second alternate protocol describes a transfer method based on a different transfer-stack setup. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel.
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