M13 clones carrying point mutations: identification by solution hybridization.
A solution hybridization procedure for the rapid identification of M13 clones carrying a particular sequence is described. The method, which employs a radiolabeled oligonucleotide probe, can discriminate between sequences which differ by only a single base, and can therefore be used for the identification of mutant sequences created by oligonucleotide-directed mutagenesis. Samples of phage-containing supernatant from cultures of M13-infected Escherichia coli are incubated with radiolabeled probe in the presence of sodium dodecyl sulfate. The mixtures are then subjected to agarose gel electrophoresis to separate hybrid molecules from unbound probe and hybridization is detected by autoradiography. This solution hybridization procedure is quicker and more convenient than membrane hybridization and has the added advantage that more than one probe can be used on a given gel.
D000942:Antigens, Bacterial / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000032:analysis*; D012996:Solutions
D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D009841:Oligonucleotides; D012996:Solutions