Can I use In-Fusion Cloning to clone GC-rich DNA fragments?

Yes, but special consideration should be given to the homologous overlaps of adjacent DNA fragments. Since In-Fusion Cloning is based on the annealing of these overlaps, it is important to take the GC content into account for the 15-bp homology. We have no specific data showing variability of current In-Fusion Cloning master mix performance depending on the GC content of the 15-bp overlap. [...]

Can I clone an oligonucleotide/shRNA oligonucleotide using In-Fusion Cloning?

Yes, synthetic oligonucleotides (?50 bp), carrying homologous overlaps with the termini of the linear vector, can be cloned using In?Fusion Cloning. For cloning of short synthetic oligos (between 50 bp and 150 bp), the suggested oligo-to-vector molar ratio is 5–15:1. Depending on the oligo length, the optimal molar ratio must be determined empirically. Note: Non-phosphorylated oligonucleotides [...]

Can I use In-Fusion Cloning for mutagenesis?

Yes, In-Fusion Cloning allows single or multiple base changes, deletions, and insertions. For details, please see the Mutagenesis with In-Fusion Cloning tech note.In-Fusion Cloning [...]

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.In-Fusion Cloning [...]

Can I use In-Fusion Cloning to clone a DNA fragment generated by restriction digest?

Yes—if the adjacent DNA fragments/oligos or linearized vector carry the 15-bp homologous overlaps required for annealing. 15-bp overlaps with the digested cloning insert may be added to the termini of a PCR-linearized vector or a synthetic oligonucleotide.In-Fusion Cloning [...]