Synthetic DNA pp 205-214

Part of the Methods in Molecular Biology book series (MIMB, volume 1472)

Rapid Construction of Recombinant Plasmids by QuickStep-Cloning

Protocol

Abstract

QuickStep-Cloning is a novel molecular cloning technique that builds upon the concepts of asymmetric PCR and megaprimer-based amplification of whole plasmid. It was designed specifically to address the major drawbacks of previously reported cloning methods. The fully optimized protocol allows for a seamless integration of a long DNA fragment into any position within a plasmid of choice, in a time-efficient and cost-effective manner, without the need of a tedious DNA gel purification, a restriction digestion, and an enzymatic ligation. QuickStep-Cloning can be completed in less than 6 h, significantly faster than most of the existing cloning methods, while retaining high efficiency.

Key words

Recombinant DNA Megaprimer PCR Ligation-independent cloning Plasmid construction Synthetic biology Protein engineering Metabolic engineering 

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.ChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological EngineeringUniversity of SheffieldSheffieldUK

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