What are the considerations for the location of homologous overlaps?
What are the considerations for the location of homologous overlaps?
The 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 locus, regardless of available restriction sites, thus enabling the generation of the 15-bp overlaps at the termini at a particular position.