1/29/2024 0 Comments Steps of northern blotting![]() ![]() ![]() 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. The Southern blotting technique was named after Edwin Southern who introduced the technique in 1975. It is a multi-step process, that begins with the electrophoresis of DNA, transfer of DNA fragments into nitrocellulose strip, and exposing those fragments to a DNA probe labeled with a radioactive or chemical tag. Southern blotting is a molecular technique to find target DNA sequences in a sample. ![]()
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