Can I split the homologous 15-nt overlap between the insert and vector, or adjacent inserts?

Yes. The homologous 15-nt overlap can be split between adjacent DNA fragments. However, splitting the overlap between an insert and vector can only be done if the vector is linearized via inverse PCR. Primer design for this option is not facilitated by the online Primer Design Tool.In-Fusion Cloning [...]

Can I clone multiple fragments into one vector in a single In-Fusion Cloning reaction?

Yes—we have successfully tested multiple-fragment cloning with up to five inserts (see the figure and table below for cloning schematic and colony screen results, respectively). Primer design for multiple-fragment cloning can be done with our online Primer Design Tool. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer.) [...]

Do PCR-generated 3' A-overhangs interfere with In-Fusion Cloning?

No, 3' A-overhangs do not interfere with the In?Fusion Cloning mechanismIn-Fusion Cloning [...]

What PCR polymerases are recommended for amplification of the In-Fusion cloning insert?

In-Fusion Cloning is compatible with any PCR polymerase. 3' overhangs do not interfere with the cloning reaction. To ensure an error-free insert, use a polymerase with high proofreading activity, like PrimeSTAR Max DNA Polymerase (supplied with some In?Fusion Snap Assembly bundles). This polymerase is highly robust and accurate, enabling amplification of up to 6 kb of human genomic DNA, 10 kb of [...]

What oligonucleotide quality is required for an In-Fusion PCR primer?

In-Fusion PCR primers should be high-quality oligonucleotides, purified by desalting. Gel or HPLC purification is not required.In-Fusion Cloning [...]