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Conservative Site-specific Recombination and Phase Variation02:53

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Cloning in Plasmid Vectors: Directional Cloning.

Michael R Green, Joseph Sambrook

    Cold Spring Harbor Protocols
    |November 3, 2020
    PubMed
    Summary

    This protocol details a reliable method for cloning linear DNA fragments with incompatible ends. It ensures successful insertion into a linearized vector, a fundamental molecular biology technique.

    Area of Science:

    • Molecular Biology
    • Genetic Engineering

    Background:

    • Standard cloning techniques often face challenges with DNA fragments possessing incompatible ends.
    • Efficiently joining DNA fragments with non-homologous ends is crucial for various genetic manipulation applications.

    Purpose of the Study:

    • To describe a robust and established protocol for the molecular cloning of linear DNA fragments.
    • To provide a reliable method for ligating DNA fragments with incompatible termini into a linearized vector.

    Main Methods:

    • The protocol involves preparing linear DNA fragments with specific end modifications.
    • Ligation of these fragments into a correspondingly prepared linearized vector is performed.
    • Transformation and selection of recombinant clones are standard downstream procedures.

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    Main Results:

    • The described method reliably achieves the cloning of linear DNA fragments.
    • Successful ligation is demonstrated even when fragment ends are not directly compatible.
    • This technique offers a dependable solution for constructing recombinant DNA molecules.

    Conclusions:

    • This protocol provides a valuable and time-tested procedure for DNA fragment cloning.
    • It remains a relevant method for researchers needing to clone DNA fragments with incompatible ends.
    • The reliability of this approach supports its continued use in molecular biology laboratories.