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An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids.

Benoit, Roger and Wilhelm, Ramon and Scherer-Becker, Daniela and Ostermeier, Christian (2006) An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. Protein Expression and Purification, 45 (1). pp. 66-71. ISSN 1046-5928

Abstract

We describe an improved, universal method for the seamless integration of DNA fragments into plasmids at any desired position. The protocol allows in vitro joining of insert and linearized plasmid at terminal homology regions using the BD In-Fusion cloning system. According to the standard BD In-Fusion protocol, vectors are linearized by restriction enzyme digestion. Linearization of plasmids by polymerase chain reaction (PCR), instead of restriction enzyme digestion, extends the usefulness of the method by rendering it independent of restriction endonuclease recognition sites and by allowing seamless insertion of DNA fragments at any position, without introduction of unwanted nucleotides flanking the site of insertion. The combination of PCR linearization of plasmids and BD In-Fusion technology has shown to be very useful for the insertion of genes into the expression regions of multiple plasmids for the heterologous expression of proteins in Escherichia coli. Hands-on time is minimal and there is no need for preparative gel electrophoresis. The protocol is very simple and only involves PCR and liquid handling steps. The method should therefore theoretically have a good potential for automation.

Item Type: Article
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Additional Information: author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords: In-Fusion; Homologous recombination; Heterologous protein expression; Seamless cloning
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Date Deposited: 14 Dec 2009 13:59
Last Modified: 31 Jan 2013 01:16
URI: https://oak.novartis.com/id/eprint/478

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