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