The Southern blotting technique involves transfer of DNA fragments separated in electrophoretic gels to membrane filters for detection of specific base sequences by complementary probes. Southern blots are used to identify and quantitate specific DNA sequences.

Restriction fragment length polymorphism (RFLP) refers to the variation among individuals in the lengths of DNA fragments produced by restriction enzymes that cut DNA at specific sites. The variations are due to differences in the DNA sequence at these sites from person to person. Analysis of RFLPs is used in the construction physical maps of the genome, genetic linkage maps, and for forensic testing. RFLPs are normally determined and assayed by Southern blot.

1. Inappropriate membrane was used.
2. Immobilization of nucleic acids to membrane was not efficient.
3. Probe concentration was too low.
4. Hybridization and washing conditions were too stringent.

This is the most common problem with Southern blots.

1. Concentration of probe was too high.
2. Hybridization and washing conditions were not appropriate.
3. Blocking was inadequate.
4. Washing was inadequate.
5. Target concentration was too high.
6. There was non-specific binding of detection reagents to the membrane.

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Price, D.C. (1996) Chemiluminescent substrates for detection of restriction fragment length polymorphism. Sci. Justice 36(4): 275-82.

Sambrook, J., Fritsch, E.M., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.