How do I generate homologous overlaps between the termini of cloning inserts and linearized vectors?

Homologous inserts are created through PCR amplification of cloning inserts using primers specifically designed to incorporate 15 nt of 5' overhangs complementary to the termini of the linearized vector (or adjacent cloning insert). Alternatively, 15 nt of homology may be added to a vector linearized via inverse PCR, such that it overlaps with the cloning insert. If synthetic oligonucleotides [...]

What are the requirements for a homologous overlap that will facilitate a successful In-Fusion Cloning reaction?

Homologous overlaps are necessary for In?Fusion Cloning. Appropriate homology consists of a 15-nt DNA sequence complementary to the 5' end of a linearized cloning vector or cloning insert. For multiple-insert cloning, we recommend increasing homology to 20 nt. Figures 2 and 3 in the In?Fusion Snap Assembly User Manual or In?Fusion Snap Assembly EcoDry User Manual provide detailed [...]

How does In-Fusion Snap Assembly compare to NEBuilder HiFi DNA Assembly?

In-Fusion Snap Assembly produces more colonies than NEBuilder HiFi for most cloning experiments (e.g., single-insert cloning, large-vector cloning, and site-directed mutagenesis) and always yields a cloning accuracy >90%. For multiple-insert cloning, In-Fusion Snap Assembly only requires a 15-minute incubation, compared with up to 60 minutes for NEBuilder HiFi.In-Fusion Cloning [...]

In-Fusion HD and In-Fusion Snap Assembly bundles include different PCR polymerases. Should I expect a difference in performance?

No. While the In-Fusion HD bundles include CloneAmp HiFi PCR Premix and In-Fusion Snap Assembly bundles include PrimeSTAR Max PCR Premix, both enzymes provide exceptionally high fidelity and will give you the same trusted results.In-Fusion Cloning [...]

What is the difference between In-Fusion Snap Assembly and In-Fusion HD Cloning?

In-Fusion Snap Assembly offers all the same benefits and uses the same streamlined protocol as In-Fusion HD Cloning, but yields higher efficiency (number of colonies) and consistency, especially with challenging projects like multiple-fragment cloning.In-Fusion Cloning [...]