Abstract
Ligation-independent cloning (LIC) is a simple, rapid, and efficient method for high-throughput cloning. In this system, linear plasmid vector and insert DNA are treated to generate complementary single-stranded overhangs that anneal during a short incubation.
The LIC system is adaptable for use with any vector following an alteration of the vector sequence. This chapter describes the creation of an LIC-compatible vector, with tips on how to make any vector LIC-enabled. It also includes a protocol for generating high-quality linearized vector template for the LIC reaction. Lastly, a step-by-step protocol of the LIC reaction is outlined, with useful tips and tricks for optimization and screening.
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References
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Acknowledgments
The author thanks Michael Murphy, Peter Beernink, and Paul Richardson for helpful suggestions and critical reading of the manuscript. This work was performed under the auspices of the US Department of Energy, Office of Biological and Environmental Research, by the University of California, Lawrence Livermore National Laboratory (contract No. W-7405-Eng-48), Lawrence Berkeley National Laboratory (contract No. DE-AC03-76SF00098), and Los Alamos National Laboratory (contract No. W-7405-ENG-36).
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© 2005 Humana Press Inc.
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Doyle, S.A. (2005). High-Throughput Cloning for Proteomics Research. In: Zanders, E.D. (eds) Chemical Genomics. Methods in Molecular Biology™, vol 310. Humana Press. https://doi.org/10.1007/978-1-59259-948-6_7
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DOI: https://doi.org/10.1007/978-1-59259-948-6_7
Publisher Name: Humana Press
Print ISBN: 978-1-58829-399-2
Online ISBN: 978-1-59259-948-6
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