Do I need to use phosphorylated PCR primers for In-Fusion Cloning?

No—the use of phosphorylated oligonucleotides is not required for In?Fusion Cloning.In-Fusion Cloning [...]

For In-Fusion Cloning, is it a problem if the 15-bp region of homology is present more than once in the vector? Will multiple recombination products result?

Internal recombination events at sites other than those adjacent to the vector linearization site are extremely rare. Therefore, even if your desired region of homology is present more than once in the vector sequence, unwanted recombination events are unlikely to occur.In-Fusion Cloning [...]

Can small external sequences be included in the In-Fusion PCR primer?

Yes—external nucleotide sequences (e.g., small tags, Kozak sequences, restriction sites, etc.) can be added between the template/gene-specific portion and the 15-nt homologous overlap of the In?Fusion PCR primer.In-Fusion Cloning [...]

How do I clone my gene of interest in the same translational reading frame as a tag present in the cloning vector (e.g., fluorescent protein, Myc, HA, etc.)?

Translational reading frame continuity with a tag is adjusted within the length of the gene-specific portion of the PCR primer, or by adding nucleotides between the gene-specific portion and the 15-nt homology of the PCR primer. Please note that the current version of our online Primer Design Tool does not allow an adjustment for translational reading frame continuity. As such, the primer [...]

How can I alter the reading frame when performing In-Fusion Cloning?

The reading frame is defined by the primer sequence. For example, when creating a fusion protein, if the 15 bp of vector homology at the 5' end of the In?Fusion PCR primer sequence corresponds to the last five codons of the vector reading frame, you would clone your new gene or tag in the same reading frame downstream of the C-terminus of the vector sequence by placing the first codon of this [...]