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        Self-circularization of Linear DNA

        互联网

        766

         

         

        1. In a microcentrifuge tube prepare a solution of linear DNA (25-50ng) in deionized water or TE buffer (10-35µl).
        2. Add:
          • 10X ligation buffer 5µl,
          • 50% PEG 4000 solution (for blunt ends only)  5µl,
          • deionized water to 50µl,
          • T4 DNA Ligase 5u.
            Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.
        3. Incubate the mixture for 1 hour at 22°C .
        4. Inactivate T4 DNA Ligase by heating reaction mixture at 65°C for 10 minutes.
        5. Resulting reaction mixture can be used directly for transformation.

        References

        1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
        2. Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.

         

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