What are the considerations for the location of homologous overlaps?

The homologous 15-bp overlaps should be located precisely at the termini of the vector and insert. 15-bp complementary regions not located at the termini of adjacent DNA fragments will not be joined by In?Fusion Cloning. PCR linearization of a vector allows positioning of the primers at the desired cloning locus, regardless of available restriction sites, thus enabling the generation of the [...]

What's the optimal incubation time for In-Fusion?

An increase in the In-Fusion reaction time is not recommended. It may generate uneven single-stranded regions at the ends of the cloning insert and vector, resulting in inefficient annealing of the homologous overlaps, thus reducing cloning efficiency.In-Fusion Cloning [...]

How should I set up control reactions for In-Fusion?

Always perform a control In?Fusion Cloning reaction using the control vector (linearized pUC19) and control insert provided in each kit. If your experiment produces unexpected results, the control reaction can help you to determine where to start troubleshooting. The negative control provided with the kit typically produces fewer than 5% blue colonies; the number of white colonies produced [...]

What's the molar ratios between vector and insert for In-Fusion?

In-Fusion Cloning uses a very robust enzyme, and allows highly efficient cloning in most situations. General recommendations on insert/vector quantities are included in all current In-Fusion Cloning user manuals. 1. To ensure optimal results under standard conditions, or when performing single- or multiple-fragment cloning, use an insert-to-vector ratio of 2:1. -The molar ratio of each of the [...]

Is In-Fusion capable of working with site-directed mutagenesis?

In-Fusion Cloning allows single or multiple base changes, deletions, and insertions. For details, please see our Mutagenesis with In-Fusion Cloning tech note.In-Fusion Cloning [...]