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    Propagating Gateway Vectors

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    Authors
    Reece-Hoyes, John S.
    Walhout, Albertha J. M.
    UMass Chan Affiliations
    Program in Molecular Medicine
    Program in Systems Biology
    Document Type
    Journal Article
    Publication Date
    2018-01-02
    Keywords
    Genetic Phenomena
    Genetics and Genomics
    Genetic Structures
    Investigative Techniques
    Laboratory and Basic Science Research
    Molecular Biology
    Systems Biology
    
    Metadata
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    Link to Full Text
    https://doi.org/10.1101/pdb.prot094920
    Abstract
    Generating stocks of Entry and Destination vectors for use in the Gateway recombinatorial cloning system requires transforming them into Escherichia coli strain DB3.1, where they can replicate because this strain is immune to the effects of the ccdB gene carried in the Gateway cassette. However, mutations in the ccdB gene can arise at low frequency, and these mutant plasmids will consequently allow growth of standard cloning strains of E. coli (e.g., DH5alpha). Therefore, after making new stocks of Gateway plasmids, their ability to grow in cloning strains of E. coli must be tested. This involves obtaining multiple stocks of vector, each arising from a single plasmid grown in a single DB3.1 bacterial colony, and transforming each stock into both DB3.1 and the preferred cloning strain of E. coli in a controlled fashion. Only vector stocks that effectively kill the standard cloning strain (i.e., no or few colonies are obtained after transformation) should be used in Gateway cloning reactions. The sequence can be performed in 3 d.
    Source

    Cold Spring Harb Protoc. 2018 Jan 2;2018(1):pdb.prot094920. doi: 10.1101/pdb.prot094920. Link to article on publisher's site

    DOI
    10.1101/pdb.prot094920
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/49849
    PubMed ID
    29295904
    Related Resources

    Link to Article in PubMed

    ae974a485f413a2113503eed53cd6c53
    10.1101/pdb.prot094920
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