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Rapid Construction of Recombinant Plasmids by QuickStep-Cloning

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

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

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  • DOI: 10.1007/978-1-4939-6343-0_16
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References

  1. Tee KL, Wong TS (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnol Adv 31:1707–1721

    CAS  CrossRef  PubMed  Google Scholar 

  2. van den Ent F, Lowe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74

    CrossRef  PubMed  Google Scholar 

  3. Miyazaki K (2011) MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids. Methods Enzymol 498:399–406

    CAS  CrossRef  PubMed  Google Scholar 

  4. Jajesniak P, Wong TS (2015) QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids. J Biol Eng 9:15

    CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    CAS  CrossRef  PubMed  Google Scholar 

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Correspondence to Tuck Seng Wong .

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Jajesniak, P., Wong, T.S. (2017). Rapid Construction of Recombinant Plasmids by QuickStep-Cloning. In: Hughes, R. (eds) Synthetic DNA. Methods in Molecular Biology, vol 1472. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6343-0_16

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  • DOI: https://doi.org/10.1007/978-1-4939-6343-0_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6341-6

  • Online ISBN: 978-1-4939-6343-0

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