DNA Cloning and Assembly Methods pp 59-72

Part of the Methods in Molecular Biology book series (MIMB, volume 1116) | Cite as

USER-Derived Cloning Methods and Their Primer Design

  • Bo Salomonsen
  • Uffe H. Mortensen
  • Barbara A. Halkier
Protocol

Abstract

Uracil excision-based cloning through USER™ (Uracil-Specific Excision Reagent) is an efficient ligase-free cloning technique that comprises USER cloning, USER fusion, and USER cassette-free (UCF) USER fusion. These USER-derived cloning techniques enable seamless assembly of multiple DNA fragments in one construct. Though governed by a few simple rules primer design for USER-based fusion of PCR fragments can prove time-consuming for inexperienced users. The Primer Help for USER (PHUSER) software is an easy-to-use primer design tool for USER-based methods. In this chapter, we present a PHUSER software protocol for designing primers for USER-derived cloning techniques.

Key words

Primer design USER fusion PCR-based cloning Seamless DNA fusion Ligation-free cloning PHUSER 

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Bo Salomonsen
    • 1
  • Uffe H. Mortensen
    • 2
  • Barbara A. Halkier
    • 1
  1. 1.DynaMo Center of Excellence, Department of Plant and Environmental Sciences, Faculty of ScienceUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of Systems Biology, Center for Microbial BiotechnologyTechnical University of Denmark (DTU)Kongens LyngbyDenmark

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