Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands.
We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.
D000818:Animals; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics*; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors; D051379:Mice / Q000235:genetics; D009693:Nucleic Acid Hybridization; D012343:RNA, Transfer / Q000235:genetics*
D004274:DNA, Recombinant; D004279:DNA, Viral; D012343:RNA, Transfer