How should I generate homologous overlaps of DNA fragments?
How should I generate homologous overlaps of DNA fragments?
15-bp homologous overlaps between cloning termini facilitate all In-Fusion Cloning reactions. They can be generated in the following ways:
-PCR amplification of a cloning insert using PCR primers carrying 15-nt 5' overhangs that are homologous to the termini of the linearized vector or adjacent insert
-PCR linearization of the destination vector, using PCR primers carrying 15-nt 5' overhangs homologous with the cloning insert(s)
-Oligonucleotide synthesis, generating 15-nt 5' end overhangs homologous with the termini of the linearized vector or adjacent insert. High-quality, non-phosphorylated oligonucleotides purified by desalting are compatible with In?Fusion Cloning. Gel or HPLC purification of oligonucleotides is not required.