Oct 12, 2013

Golden GATEway cloning--a combinatorial approach to generate fusion and recombination constructs

PloS One
Stephan KirchmaierJoachim Wittbrodt

Abstract

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway(TM) cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway(TM) cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable...Continue Reading

  • References16
  • Citations18

References

Mentioned in this Paper

Embryo
Beta-Actin
Shuttle Vectors
Polymerase
Untranslated Regions
Dimethyl Sulfoxide
Enzymes, antithrombotic
expression vector
Genes, Spliced
Mutagenesis, Site-Directed

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