Will In-Fusion technology allow cloning of an insert if the sites of complementarity are located at a distance from the linearized vector termini?
Will In-Fusion technology allow cloning of an insert if the sites of complementarity are located at a distance from the linearized vector termini?
No—homologous 15-bp overlaps should be located precisely at the termini of the vector and insert. 15-bp complementary regions not located at the termini of adjacent DNA fragments will not be joined by In-Fusion Cloning. PCR linearization of a vector allows positioning of the primers at the desired cloning site, thus enabling generation of the 15-bp overlaps at the termini.