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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




Journal article


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

Publication Date





75 - 90


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


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