Gibson Assembly Calculator

Gibson Assembly is a molecular biology technique for joining DNA fragments together without restriction enzymes — this Gibson Assembly Calculator takes the guesswork out of setting up your reaction. Enter your vector size, insert size, vector amount, insert:vector molar ratio, and overlap length to get the exact insert amount required in ng. Secondary outputs include total DNA, vector and insert pmol values, and the water volume needed to complete your reaction.

bp

Size of linearized vector backbone in base pairs

bp

Size of DNA fragment to insert

ng

Recommended: 50 ng of linearized vector

Recommended: 2-3× molar excess of insert

bp

Optimal: 15-40 bp (20-25 bp recommended)

Results

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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Frequently Asked Questions

What is Gibson Assembly?

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.

What is the optimal insert to vector molar ratio?

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.

How long should the overlap regions be?

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.

How much vector DNA should I use per reaction?

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.

Can Gibson Assembly join more than two fragments?

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.

What temperature and time should I use for Gibson Assembly?

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.

Why is my Gibson Assembly efficiency low?

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.

Do I need to phosphorylate DNA ends for Gibson Assembly?

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.