In-Fusion Cloning
Course Content
In-Fusion Cloning with SnapGene
In-Fusion Cloning with Multiple Inserts
Common Questions About Using SnapGene
What does SnapGene's In-Fusion cloning simulation cover?
The simulation designs PCR primers with overlapping ends that match the linearised vector, sets the overlap length and Tm, and then generates a product file. In the Product tab, Sequence view shows the fusion boundaries so you can verify in-frame fusions. The History view records all simulated steps and primers created during the procedure.
Can SnapGene retain or remove restriction sites when designing In-Fusion primers?
Yes. In the Product tab, you can check options to either regenerate or remove the partial restriction sites at the ends of the linearised vector before designing primers. This is useful when the same restriction sites need to be preserved for future cloning steps.
Can SnapGene simulate In-Fusion cloning with more than one insert?
Yes. The second video in this series covers multi-insert In-Fusion cloning. Each fragment is defined separately with its own overlap and primer settings. Sequence view in the Product tab allows you to check each fusion boundary in turn and confirm reading frame continuity across all junctions.
How does In-Fusion cloning simulation in SnapGene differ from Gibson Assembly simulation?
Both methods use overlapping ends and follow similar workflows in SnapGene. The key distinction is the enzyme system: In-Fusion uses Takara Bio's proprietary Clonase enzyme, while Gibson Assembly uses a three-enzyme mix (exonuclease, polymerase, ligase). The tools are accessed separately under Actions, and the overlap length requirements may differ between the two methods.
What overlap length should I use for In-Fusion cloning in SnapGene?
SnapGene allows you to set the overlap length when designing primers. Takara Bio recommends 15-base overlaps as standard for In-Fusion cloning, which SnapGene uses as a typical starting point. You can adjust this and the target Tm within the Product tab before designing primers.
What is the difference between this simulation series and the In-Fusion Cloning topic guide?
The simulation series shows how to run In-Fusion cloning in SnapGene step by step, from primer design through to a verified product file. The In-Fusion Cloning topic guide covers the technique itself: how the Clonase enzyme creates seamless junctions, its advantages over restriction cloning, and key considerations for primer and vector design.
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