Colony Hybridization

Colony hybridization involves, first, the cutting up with appropriate restriction endonucleases, of isolated DNA into fragments. A library of the fragments is then established by cloning the fragments with the aid of a cloning vector (e.g. cosmid, YAC, etc.). The bacteria, presumably each carrying the DNA fragments in a chimeric plasmid, are seeded at low density on agar plates so each colony would be separate. After the colonies are formed a replica plate is established from the master plate by pressing over it a membrane filter. After denaturation of the DNA on the nitrocellulose filter, it is hybridized with a labeled DNA or RNA probe. After washing off the unbound probe, the filter is autoradiographed and the colonies containing the desired molecules of DNA are identified. Since the position of the colonies corresponding to the black dots on the photographic film (dot blot) can be identified, the bacteria containing the vector with that specific DNA can be further propagated (see...