Application of the Restriction-Free (RF) Cloning for Multicomponents Assembly

  • Yoav Peleg
  • Tamar Unger
Part of the Methods in Molecular Biology book series (MIMB, volume 1116)


Molecular manipulations, including DNA cloning and mutagenesis, are currently employed on a routine basis in all life science disciplines. Over the last decade new methodologies have emerged that expanded and facilitated the applications for DNA cloning. The classical Ligation-Dependent Cloning (LDC) is gradually replaced by Ligation-Independent Cloning (LIC) techniques. The Restriction-Free (RF) cloning was originally developed for introduction of a foreign DNA into a plasmid at any desired position. The RF methodology is based on generation of a PCR product, which serves as a set of mega-primers for subsequent incorporation into any desired position within a circular plasmid. We have expanded the applications of the RF methodology for multiple simultaneous alterations of a target DNA and for multicomponents assembly. In the current manuscript we describe a step-by-step protocol for application of the RF methodology for simultaneous multiple DNA fragments assembly in tandem and at distinct positions within an expression vector.

Key words

DNA cloning Restriction-Free (RF) cloning Ligation-Independent Cloning (LIC) Multicomponents assembly 



We thank Prof. J.L. Sussman, Prof. I. Silman, Prof. G. Schreiber, and Prof. Yigal Burstein for their continuous support throughout the study. The ISPC is supported by the Divadol Foundation.


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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Yoav Peleg
    • 1
  • Tamar Unger
    • 1
  1. 1.Israel Structural Proteomics Center (ISPC), Faculty of BiochemistryWeizmann Institute of ScienceRehovotIsrael

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