NDLI: Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs

Content Provider	PubMed Central
Author	Kirchmaier, Stephan Lust, Katharina Wittbrodt, Joachim
Editor	Korzh, Vladimir
Copyright Year	2013
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 GatewayTM 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 GatewayTM cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of “classical construct generation” and allows focusing on construct design.
Related Links	http://dx.doi.org/10.1371/journal.pone.0076117
Starting Page	76117
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	10
Volume Number	8
Language	English
Publisher	Public Library of Science
Publisher Date	2013-10-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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