(1) Denhardt, D.T. (1966) Biochem. Biophys. Res. Com. 23, 641-646
A membrane-filter technique for the detection of complementary DNA.
(2) Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition; p. 9.47-9.55, 6.12. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Denhardt''s solution is a blocking reagent for preventing the unspecific binding of nucleic acids to nitrocellulose or nylon membranes in hybridisation experiments. Denatured DNA binds to nitrocellulose membranes, but not free RNA. Pretreatment of the membrane with Denhardt''s solution prevents the binding of single stranded or (unspecific) denatured DNA. The specific ''annealing'' of denatured DNA to its complementary DNA is not inhibited. The use of Denhardt''s reagent is recommended for Northern hybridisation, hybridisation of RNA, ''single copy Southern hybridisation'' or hybridisation of DNA, immobilized on nylon membranes (2).
Denhardt''s solution is set up as a 50X stock solution (50X reagent: 5 g Ficoll® 400, 5 g polyvinylpyrrolidone, 5 g BSA fraction V in water ad 500 ml), filtered and stored at -20°C. This stock solution is diluted tenfold with prehybridisation buffer (usually 6X SSC or 6X SSPE with 0.5 % SDS and 100 μg/ml denatured, sonified salmon sperm DNA; ref. 2).