Format

Send to

Choose Destination
J Struct Biol. 2010 Oct;172(1):34-44. doi: 10.1016/j.jsb.2010.06.016. Epub 2010 Jun 25.

Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression.

Author information

1
Department of Structural Biology, The Israel Structural Proteomics Center (ISPC), Weizmann Institute of Science, Rehovot, Israel. tamar.unger@weizmann.ac.il

Abstract

Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system.

PMID:
20600952
DOI:
10.1016/j.jsb.2010.06.016
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center