Directional cloning of blunt-ended PCR products.
A method that allows the directional cloning of blunt-ended polymerase chain reaction (PCR) fragments is described. One PCR primer must be 5' phosphorylated. Extra bases are not required on either PCR primer. A linearized vector is enzymatically processed to contain a single 5'-terminal phosphate. The monophosphorylated vector is amenable to recombinant-insertion during ligation when the fragment is in the correct orientation. Increased recombinant yield results from incubating the monophosphorylated vector with a restriction enzyme (SrfI) that relinearizes nonrecombinant plasmids during the ligation reaction.
Weiner
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Stratagene Cloning Systems, La Jolla, CA 92037.
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014169:Transformation, Bacterial; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000235:genetics
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase