![]() ![]() The time required for hybridization usually 1–16 hours depending on factors like the complexity of the probe and concentration. Hybridization is usually carried out in a sealed bag which will contain the Southern blot, and hybridization fluid containing the labeled probe. ![]() After incubating the prehybridization solution at 42☌, the heat snap chilled salmon sperm DNA is added to it at a concentration of 50 µg/mL. In this step, the membrane is incubated in Denhardt’s solution for 1 hour or more, depending on the type of reaction. There are various kinds of blocking agents, commonly, salmon or herring sperm DNA is used for blocking the membrane surface and target DNA. Prehybridization and blocking are done to eliminate non-specific reactions. The membrane is then baked in a vacuum or regular oven at 80 ☌ for 2 hours or can be fixed via UV crosslinking mediated by exposure to short-wavelength UV light to permanently attach the transferred DNA to the membrane. The portion of the nitrocellulose membrane, touching the gel should be gently removed using a blade. Fragments are pulled towards the nitrocellulose filter membrane by capillary action and result in the formation of an imprint or blot of the gel. A sheet of nitrocellulose membrane is placed on top of (or below, depending on the direction of the transfer) the gel and gentle 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. The process is done by either electroblotting or capillary blotting. Although this step determines the name of this technique “Southern blotting,” the term is typically used to describe the entire procedure. DNA thus obtained are double-stranded, and is therefore denatured to single-stranded DNA by dipping the gel in an alkaline solution.īlotting refers to the transfer of the fragmented DNA sequence to the nitrocellulose membrane or nylon membrane. Acrylamide gels can alternatively be used for good resolution of smaller DNA fragments (<800 bp). (Image source: National Human Genome Research Institute) Extraction, purification, fragmentation, and separation of target DNAĭNA is extracted from the target source and is broken into small fragments using restriction endonuclease enzyme.įragmented DNA is then electrophoresed on an agarose gel to separate the fragments according to their molecular weights. ![]()
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