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In this chapter, protocols for the construction of expression vectors using In-Fusion PCR cloning are presented. The method enables vector and insert DNA sequences to be seamlessly joined in a ligation-independent reaction. This property of the In-Fusion process has been exploited in the design of a suite of multi-host compatible vectors for the expression of proteins with precisely engineered His-tags. Vector preparation, PCR amplification of the sequence to be cloned and the procedure for inserting the PCR product into the vector by In-Fusion are described.

Original publication

DOI

10.1007/978-1-59745-196-3_5

Type

Journal article

Journal

Methods in molecular biology (Clifton, N.J.)

Publication Date

01/2009

Volume

498

Pages

75 - 90

Addresses

Oxford Protein Production Facility, Welcome Trust Centre for Human Genetics, Oxford, UK.

Keywords

Animals, Escherichia coli, Glycerol, Histidine, Recombinant Fusion Proteins, Cell Culture Techniques, Cloning, Molecular, Protein Engineering, Polymerase Chain Reaction, Transformation, Genetic, Genetic Vectors, Plasmids