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. DNA loading buffer, TBE Buffer for electrophoresis Steps involved in Southern blotting Southern blotting technique.Enzymes: restriction nucleases, RNase A.Cellulose nitrate or nitrocellulose membrane filter with uniform porosity.The probe also has a radioactive atom or a fluorescent dye label, that following hybridization, permits the DNA fragment of interest to be detected from different DNA fragments present on the membrane. The membrane is then treated with a small piece of DNA or RNA called a probe, which has a complementary sequence to the target DNA. Following separation, the double-stranded pieces of DNA are denatured into single strands within the gel and transferred from the gel onto a blotting membrane. The target DNA is broken into small fragments using restriction endonucleases and is separated by electrophoresis. Extraction, purification, fragmentation, and separation of target DNA.
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