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Southern Blot Protocol

   attached      bands      bind      bound      complementary      digested      emits      expose      form      fragments      incubated      labeled      location      pattern      probe      radioactive      sheet      smear      source      strands      substrate  

DNA (genomic or other ) is with a restriction enzyme and separated by gel electrophoresis, usually an agarose gel. Because there are so many different restriction fragments on the gel, it usually appears as a rather than discrete . The DNA is denatured into single by incubation with NaOH.

The DNA is transferred to a membrane which is a of special blotting paper. The DNA retain the same of separation they had on the gel.

The blot is with many copies of a made of single-stranded DNA. This can form base pairs with its DNA sequence and to a double-stranded DNA molecule. The probe cannot be seen but it is either or has an enzyme to it (e.g. alkaline phosphatase or horseradish peroxidase).

The of the probe is revealed by incubating it with a colorless that the enzyme converts to a colored product that can be seen or light which will X-ray film. If the probe was with radioactivity, it can expose X-ray film directly.