ligation calculator

How this ligation calculator works

This calculator helps you build a basic cloning ligation reaction by converting a target molar ratio into real pipetting numbers. In ligation, what matters most is molecules of insert and vector, not just mass. Since larger DNA fragments weigh more per molecule, the calculator adjusts insert mass based on fragment size.

You enter vector size, insert size, concentrations, and a desired insert:vector molar ratio (for example 3:1). The tool then computes:

• Vector and insert amounts in ng and estimated fmol
• Volumes of vector and insert to pipette
• 10× buffer volume for a 1× final concentration
• Water needed to reach your target total volume

Formula used for insert DNA mass

A common quick formula for one insert and one vector is:

Insert ng = Vector ng × (Insert bp / Vector bp) × (Insert:Vector molar ratio)

This is mathematically equivalent to molar balancing and is widely used for routine sticky-end cloning. For blunt-end ligations or difficult assemblies, you may need more optimization.

Recommended starting settings

Typical first attempt

• Vector: 25–100 ng
• Insert:vector ratio: 2:1 to 5:1
• Total reaction volume: 10–20 µL
• T4 DNA ligase: 0.5–1 µL (follow your enzyme datasheet)
• Incubation: 10 min (quick ligase) to overnight at 16°C (standard ligase workflows)

When to change the ratio

• Low colony count: try increasing insert:vector ratio to 4:1 or 5:1
• High background of empty vector: reduce vector mass, verify dephosphorylation, and gel-purify
• Very small insert (<200 bp): ratios may need extra tuning

Troubleshooting poor ligation results

• Check that both DNA concentrations are measured accurately.
• Confirm your vector is fully linearized and purified from uncut plasmid.
• Use fresh ligase buffer (ATP degrades with freeze-thaw and age).
• Minimize salts and contaminants from PCR cleanup or gel extraction.
• Always include controls: vector-only and positive ligation control if possible.

Notes and limitations

This tool is intended for educational and planning use. Multi-fragment ligations (such as 2+ inserts), Gibson assembly, Golden Gate cloning, and specialized kits use different stoichiometry and should be calculated separately. Always prioritize your enzyme manufacturer protocol over any generic calculator.