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Synthetic DNA

Volume 1472 of the series Methods in Molecular Biology pp 205-214

Date:

Rapid Construction of Recombinant Plasmids by QuickStep-Cloning

  • Pawel JajesniakAffiliated withChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, University of Sheffield
  • , Tuck Seng WongAffiliated withChELSI Institute and Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, University of Sheffield Email author 

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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