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        Linker Ligation

        互联网

        1160

         

        Linker Ligation (with T4 DNA Ligase )

        1. In a microcentrifuge tube prepare a solution of blunt ended, dephosphorylated DNA (100-500ng) in TE buffer (5-7µl).
        2. Add 1-2µg of phosphorylated linkers in 5µl of TE buffer.
        3. Add:
          • 10X ligation buffer 2µl,
          • 50% PEG 4000 solution   2µl,
          • deionized water to 20µl,
          • T4 DNA Ligase 2u.
            Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.
        4. Incubate the mixture for 1 hour at 22°C .
        5. Inactivate T4 DNA Ligase by heating the reaction mixture at 65°C for 10 minutes. The resulting ligation products can be digested directly with restriction endonucleases.

         

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