In-Fusion HD Cloning Plus products enable directional, seamless cloning of any PCR fragment—or multiple fragments—into any linearized vector in a single 15-minute reaction. No additional treatment of the PCR fragment (such as restriction digestion, ligation, phosphorylation, or blunt-end polishing) is required. The efficiency of In-Fusion HD Cloning Plus is over 95%, providing confidence in your clones and enabling you to scale up for high-throughput workflows.

The In-Fusion HD enzyme premix fuses PCR-generated sequences and linearized vectors efficiently and precisely, utilizing a 15-bp overlap at their ends. This 15-bp overlap can be engineered by designing custom primers for amplification of the desired sequences using our primer design tool. The In-Fusion enzyme mix removes nucleotides from the 3' end of the linear DNA strands. This allows complementary base pairs between two pieces of DNA to anneal, leading to fragment joining. This method can be used to clone single or multiple fragments into a single vector without subcloning. Additional applications include mutagenesis, gene synthesis, gene design, domain swapping and domain modification.

The ability to clone directly into any destination vector at any locus (without the use of restriction enzymes) eliminates the need for any further subcloning or manipulation, simplifying the cloning workflow and further enabling a transition to high-throughput applications.

In-Fusion HD Cloning Plus system components

All In-Fusion HD Cloning products contain the In-Fusion HD Enzyme Premix and reagents for control experiments. In-Fusion HD Cloning Plus also includes:

• In-Fusion HD Enzyme Premix (liquid; store at –20°C)

  In-Fusion HD enzyme mix fuses DNA fragments efficiently and precisely by recognizing a 15-bp overlap at their ends—without the use of ligase. Provided in a liquid format as a 5X enzyme premix in 20-μl or 100-μl aliquots, depending on kit size. Note: the EcoDry kit format includes lyophilized cloning enzyme, prealiquoted in 8-well tube strips or 96-well plates, depending on kit size.

• CloneAmp HiFi PCR Premix (liquid; store at –20°C)

  CloneAmp HiFi PCR Premix is a convenient 2X liquid master mix that offers exceptionally accurate, efficient, and fast DNA amplification. The premix contains dNTPs and an optimized buffer, allows rapid set-up of PCR reactions, and facilitates successful cloning. The polymerase master mix is provided in 625-μl aliquots in both EcoDry and liquid kits.

• Stellar Competent Cells (frozen; store at –80°C)

  High-efficiency competent cells are essential to the success of In-Fusion Cloning. An E. coli HST08 strain is included that provides high transformation efficiency (greater than 5 x 10⁸ cfu/µg) and complements the efficiency of all In-Fusion HD Cloning Plus kits. Cells are provided in 100-μl aliquots in individual tubes. The 96-rxn kit includes the cells prealiquoted in 96-well plates (20 μl in each well).

• Cloning Enhancer or NucleoSpin Gel and PCR Clean-up kit (liquid; store at –20°C or room temperature, respectively)

  Cloning Enhancer (CE) is an easy-to-use enzyme premix that removes background plasmid DNA and PCR residue, eliminating the need for PCR purification prior to cloning. In-Fusion HD Cloning Plus CE kits are ideal for cloning when there is a single PCR product on the gel, and can be used as part of a streamlined workflow for high-throughput systems. Cloning Enhancer is provided in 50-μl aliquots. Alternatively, NucleoSpin Gel and PCR Clean-up is suitable for gel extraction as well as PCR purification . Provided as individual columns or 96-well plate purifications, depending on kit size.