Brown T
University of Manchester Institute of Science and Technology, Manchester, United Kingdom.
Curr Protoc Mol Biol. 2001 May;Chapter 2:Unit2.10. doi: 10.1002/0471142727.mb0210s21.
The principle of hybridization analysis is that a single-stranded DNA or RNA molecule of defined sequence (the "probe") can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target") that has been immobilized on a nitrocellulose and nylon (uncharged and charged) membrane support. The approach taken in this unit is to present as the basic protocol an unsophisticated procedure for hybridization analysis with a radiolabeled DNA probe. The alternate protocol describes a similar method for probing DNA blots with a radiolabeled RNA probe. A support protocol for stripping blots for reprobing is also provided. The commentary describes modifications, including changes to prehybridization, hybridization, and wash solution formulations, and alterations to incubation times and conditions, the latter including a discussion of the wash conditions compatible with different degrees of stringency.
