In-Fusion Cloning in Geneious Prime

This video shows how to simulate In-Fusion cloning in Geneious Prime: linearize a vector by restriction digest or inverse PCR, then run the In-Fusion Cloning tool with a 3' exonuclease. Geneious sets a 15 bp minimum overhang for single inserts, or 20 bp for multiple inserts, and generates primers. Review the Homology Assembly Report, final construct, and generated primers.

Perform In-Fusion Cloning

Linearize a vector by restriction digest or inverse PCR, then run In-Fusion Cloning with a default 15 bp overhang for a single insert or 20 bp for multiple inserts. Review the Homology Assembly Report, final construct, and generated primers.

LENGTH 5 minutes

Common Questions about In-Fusion Cloning

What In-Fusion cloning steps can I simulate in Geneious Prime?

Geneious Prime supports In-Fusion cloning simulation across the full workflow: preparing DNA fragments, linearizing the vector at the desired insertion site, configuring the reaction parameters, and evaluating the assembled construct. The tool uses the same homology-based assembly logic as Gibson Assembly, adapted to the In-Fusion exonuclease chemistry and 15 bp overhang for a single insert or 20 bp for multiple inserts.

Can I perform batch In-Fusion cloning in Geneious Prime?

Geneious Prime supports batch cloning for homology-based methods including In-Fusion, allowing researchers to process multiple insert variants against the same linearized vector in a single run using sequence lists. This is useful for screening libraries or validating multiple constructs before proceeding to bench work.

How does Geneious Prime handle In-Fusion primer design?

Geneious Prime designs In-Fusion cloning primers with the homologous extensions required to create the 15-bp overlaps between insert and linearized vector. The tool calculates melting temperatures for both the gene-specific binding region and the homology extension, and generates primer annotations for review before synthesis. Researchers can confirm primer compatibility with their vector backbone without manual overlap calculations.

What does Geneious Prime check when simulating an In-Fusion reaction?

When simulating an In-Fusion reaction, Geneious Prime validates that overlap regions between the insert and linearized vector are correctly positioned and of sufficient length, calculates Tm for primers across both binding and extension regions, and displays the annotated final construct confirming scarless junction formation. Any issues with overlap placement or primer Tm are flagged before synthesis orders are placed.

Is In-Fusion cloning supported through the same interface as Gibson Assembly in Geneious Prime?

In-Fusion cloning and Gibson Assembly share a common interface in Geneious Prime, as both are homology-based isothermal methods. Researchers familiar with one method can apply the same workflow to the other, selecting the relevant method-specific parameters. This consistency across cloning methods reduces the learning curve for labs that use multiple ligation-independent strategies.

Does simulating In-Fusion cloning in Geneious Prime require programming?

In-Fusion cloning simulation in Geneious Prime requires no programming. The workflow is conducted entirely through the visual cloning interface — researchers select sequences, configure overlap parameters, and review the annotated construct output without writing scripts or command-line instructions. This makes the method accessible to molecular biologists at all computational levels.