How do I generate homologous overlaps between the termini of cloning inserts and linearized vectors?
How do I generate homologous overlaps between the termini of cloning inserts and linearized vectors?
Homologous inserts are created through PCR amplification of cloning inserts using primers specifically designed to incorporate 15 nt of 5' overhangs complementary to the termini of the linearized vector (or adjacent cloning insert).
Alternatively, 15 nt of homology may be added to a vector linearized via inverse PCR, such that it overlaps with the cloning insert. If synthetic oligonucleotides (?50 bp) are being cloned, these oligos may carry the 15-nt 5' overhangs homologous to the ends of the linearized cloning vector or adjacent DNA fragments. High-quality, non-phosphorylated oligos are compatible with In?Fusion Cloning.