An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids.
作者： Roger M. Benoit , Ramon N. Wilhelm , Daniela Scherer-Becker , Christian Ostermeier
DOI: 10.1016/J.PEP.2005.09.022
关键词: Restriction enzyme 、 Polymerase chain reaction 、 Restriction fragment 、 Gel electrophoresis 、 DNA 、 Computational biology 、 Plasmid 、 Restriction map 、 Biology 、 Heterologous expression 、 Molecular biology
摘要: 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 insert and linearized plasmid terminal homology regions using BD In-Fusion cloning system. According to standard protocol, vectors are by restriction enzyme digestion. Linearization polymerase chain reaction (PCR), instead digestion, extends usefulness rendering it independent endonuclease recognition sites allowing insertion position, without introduction unwanted nucleotides flanking site insertion. combination PCR linearization technology has shown be very useful genes expression multiple heterologous proteins Escherichia coli. Hands-on time is minimal there no need preparative gel electrophoresis. simple only involves liquid handling steps. should therefore theoretically have a good potential automation.
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