Enter your Vector Size (bp), Insert Size (bp), Vector Amount (ng), preferred Insert:Vector Molar Ratio, and Overlap Length (bp) into the Gibson Assembly Calculator to find the exact Insert Amount Required (ng), plus Total DNA, Vector and Insert Pmol, and Water to Add for a perfectly balanced reaction.
Insert Amount Required
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Total DNA
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Vector Pmol
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Insert Pmol
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Water to Add
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Gibson Assembly is a molecular cloning method that allows seamless joining of multiple DNA fragments with overlapping ends. It uses a mixture of exonuclease, polymerase, and ligase enzymes to create seamless assemblies without leaving scars.
The recommended insert to vector molar ratio is 2-3:1. This ensures sufficient insert DNA for efficient assembly while avoiding excess that could lead to multiple insertions or background.
Overlap regions should be 15-40 bp long, with 20-25 bp being optimal. Shorter overlaps may reduce efficiency, while longer overlaps can increase the chance of unwanted recombination.
Use approximately 50 ng of linearized vector DNA per 20 μL reaction. This amount provides enough template for efficient assembly while maintaining good transformation efficiency.
Yes, Gibson Assembly can efficiently join 2-15 DNA fragments in a single reaction. Each fragment needs overlapping ends with its neighbors, and the calculator can help determine optimal amounts for multiple fragments.
Incubate the reaction at 50°C for 15-60 minutes. Use 15 minutes for 2-3 fragments and up to 60 minutes for more complex assemblies with 4+ fragments.
Low efficiency can result from improper fragment ratios, inadequate overlap design, degraded DNA, or incorrect reaction conditions. Ensure proper molar ratios, fresh enzymes, and optimal overlap lengths of 20-25 bp.
No, Gibson Assembly does not require phosphorylated ends. The method works with blunt or sticky ends and the assembly mix contains all necessary enzymes including ligase and polynucleotide kinase activity.