How do I calculate the 15-nt overlap if the vector is linearized via restriction digest, generating a 5' or 3' overhang?

The 5' overhang of a restriction site is included in the 15-nt complementary region. The 3' overhang of a restriction site is excluded from the 15-nt complementary region. Figures 2 and 3 in the In?Fusion Snap Assembly User Manual or In?Fusion Snap Assembly EcoDry User Manual provide detailed examples. Restriction sites used for vector linearization can be preserved in the recombinant vector by [...]

Why are homologous overlaps important for In-Fusion Cloning reactions?

The mechanism for In?Fusion Cloning reactions employs a 3' exonuclease to generate single-stranded 5' overhangs at the termini of linear double-stranded DNA. These DNA fragments are then annealed via complementary 15-nt overlaps at the termini of the insert(s) and a linearized vector. The vector can be linearized by inverse PCR or restriction digest. Restriction digest can be performed with one [...]

What tools are available to assist in the design of PCR primers compatible with In-Fusion Cloning?

Instructions for designing In?Fusion PCR primers are included in all In?Fusion Cloning user manuals. Additionally, our online Primer Design Tool facilitates primer design for single- and multiple-fragment cloning and is compatible with Mozilla Firefox or Google Chrome web browsers (Internet Explorer is not compatible with the Primer Design Tool). We also recommend SnapGene Viewer as a helpful, [...]

What is the optimal length of the homologous overlap between the termini of the PCR-amplified insert and linearized cloning vector?

Current In?Fusion reaction conditions favor 15 bp of homologous overlap for single-insert cloning, and 20 bp of homologous overlap for multiple-insert cloning. We do not recommend using overlaps shorter than 12 bp or longer than 21 bp.In-Fusion Cloning [...]

How do I design PCR primers carrying 15-nt overhangs complementary to the termini of the linearized vector or adjacent insert?

Each forward (5' ? 3' sense strand) and reverse (5' ? 3' antisense strand) PCR primer should include the following: -A 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 the linearized [...]