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Application of In-Fusion™ Cloning for the Parallel Construction of E. coli Expression Vectors

  • Louise E. Bird
  • Heather Rada
  • John Flanagan
  • Jonathan M. Diprose
  • Robert J. C. Gilbert
  • Raymond J. Owens
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1116)

Abstract

In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined.

Key words

In-Fusion™ enzyme PCR cloning Single-stranded annealing Fusion tags Co-expression Periplasmic secretion Escherichia coli 

Notes

Acknowledgements

The Oxford Protein Production Facility-UK is supported by the UK Medical Research Council and the Biotechnology and Biology Research Council (MRC Grant MR/K018779/1). We thank Jo Nettleship for helpful discussions on E. coli secretion vectors.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Louise E. Bird
    • 1
    • 2
  • Heather Rada
    • 1
    • 2
  • John Flanagan
    • 2
  • Jonathan M. Diprose
    • 1
    • 2
  • Robert J. C. Gilbert
    • 2
  • Raymond J. Owens
    • 1
    • 2
  1. 1.Oxford Protein Production Facility-UKOxfordshireUK
  2. 2.Division of Structural Biology, Henry Wellcome Building for Genomic MedicineUniversity of OxfordOxfordUK

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