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 [...]

What are PCR primer design considerations for compatible with In-Fusion Cloning?

Each forward (5' - 3' sense strand) and reverse (5' - 3' antisense strand) In?Fusion Cloning PCR primer should include the following: -Template-specific (gene-specific) portion at its 3' end. To ensure specific and efficient PCR amplification, the template-specific portion of the primer should be 18–25 nt in length. -15 nt of homology at the 5' end of the primer, complementary to the termini of [...]

What's In-Fusion compatibility with multiple inserts?

We have successfully tested multiple-fragment cloning with up to five inserts. 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). Please note that between two adjacent fragments, only one homologous overlap is required for the [...]

What's In-Fusion compatibility with small inserts?

Seamless cloning is not suitable for small inserts of less than 50 bp. This is because of the exonuclease activity removing a 15–30 bp region at both ends, which may affect a significant percentage of the insert sequence. We recommend using a traditional, ligase-based cloning method for inserts smaller than 50 bp.In-Fusion Cloning [...]

What's In-Fusion compatibility with large inserts?

In-Fusion technology has been optimized for cloning large fragments. DNA inserts up to 15 kb have been successfully cloned into pUC19 using In?Fusion Cloning.In-Fusion Cloning [...]