Filter hybridization.

Abstract

This chapter describes a standard method for the hybridization of labeled DNA probes to nucleic acids bound to a nylon matrix. Filters bearing bound nucleic acids produced by Northern blotting of RNA (Chapter 39), Southern blotting of DNA (Chapter 37), and slot blotting of DNA (Chapters 35) or RNA (Chapter 40) are hybridized to labeled probes using the method described below. The advantages of this method are, first, that the use of a high concentration of SDS in the hybridization buffer ensures a low background level of nonspecific probe adherence to the membrane and, second, an extended period of filter prehybridization is not required. The inclusion of a large amount of SDS does, however, necessitate that the nucleic acids are covalently bonded to the matrix by UV light crosslinking. The inclusion of formamide (15% [v/v]) is also recommended in order to reduce the viscosity of the hybridization buffer. Formamide also has the effect of reducing the temperature of the hybridization reaction.