What is the method of Southern blotting? Why does the DNA segment get trapped in the nitrocellulose membrane?

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ncchemist eNotes educator| Certified Educator

A Southern blot is a method in microbiology to test for the presence of a particular sequence of DNA in a sample.  The DNA sample is first separated on a gel using standard gel electrophoresis.  Once the DNA fragments are separated by size, they are washed with a denaturing solution to separate the double helical strands into single strands.  The gel is then pressed against a nitrocellulose plate to transfer the DNA strands onto the plate.  The DNA binds to the nitrocellulose via electrostatic interactions (the DNA is negatively charged and the nitrocellulose is positively charged).  The nitrocellulose plate is then treated with a solution containing a marker probe.  This probe is a strand of DNA or RNA that is complementary to the particular DNA fragment being searched for.  After treatment, it is important to wash the plate with a neutral buffer solution to remove any excess probe and not get a false positive.  Finally, this plate is then scanned for any of the probe that has stuck to the plate using UV light, photographic film, or color dye, depending on the specific probe used.  If any of the probe is detected on the plate, then you know that the particular fragment of DNA that you are searching for is present on the plate and therefore in the original DNA sample that you started the whole Southern blotting process with.

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