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  • Title: Directional cloning of blunt-ended PCR products.
    Author: Weiner MP.
    Journal: Biotechniques; 1993 Sep; 15(3):502-5. PubMed ID: 8217165.
    Abstract:
    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.
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