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Assembling Linear DNA Templates for In Vitro Transcription and Translation

  • Viktor Stein
  • Miriam Kaltenbach
  • Florian HollfelderEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 815)

Abstract

Cell-free expression systems provide straightforward access from genes to the corresponding proteins, involving fewer handling steps than in vivo procedures. A quick procedure to assemble a gene of interest into a linear DNA template together with 3′- and 5′-untranslated regions using a coupled uracil-excision–ligation strategy based on USER Enzyme and T4 DNA ligase. This methodology will be useful for repeated cycles of expression and in vitro selection, in which gene libraries are repeatedly assembled and their products and templates regenerated.

Key words

Linear DNA template assembly USER friendly cloning In vitro screening and selection In vitro transcription/translation 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Viktor Stein
    • 1
  • Miriam Kaltenbach
    • 1
  • Florian Hollfelder
    • 1
    Email author
  1. 1.Department of BiochemistryUniversity of CambridgeCambridgeUK

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