Calculate how much insert to add to a ligation for a given insert:vector molar ratio. Enter the vector and insert lengths, the vector mass and your desired ratio — the tool returns the mass of insert to use.
Insert mass = ratio × vector mass × (insert length / vector length). A 3:1 insert:vector ratio is a common starting point for sticky-end ligations.
Why molar ratio, not mass
Ligation efficiency depends on the number of molecules, not their mass. Because longer fragments weigh more per molecule, you can’t just mix equal masses. The insert mass you need is:
insert mass = ratio × vector mass × (insert length / vector length)
So for a 3:1 insert:vector ratio with 50 ng of a 5 kb vector and a 1 kb insert, you need 3 × 50 × (1000/5000) = 30 ng of insert.
Which ratio should I use?
- Sticky-end (cohesive) ligations: a 3:1 insert:vector molar ratio is a common starting point; 1:1 to 5:1 all work.
- Blunt-end ligations: often need a higher ratio (5:1 or more) because they ligate less efficiently.
- Large inserts: ratios closer to 1:1 can help.
Good ligation practice
- Include a vector-only (no insert) control to gauge background from self-ligation/uncut vector.
- Dephosphorylate the vector if it self-ligates heavily.
- Keep total DNA reasonable (typically tens to ~100 ng) in the reaction.
- Confirm your sites and fragment ends first with NEBcutter, and map the final construct with PlasMapper.
Frequently asked questions
What insert:vector ratio should I use?
3:1 is a typical starting point for sticky-end ligations; blunt-end ligations often use 5:1 or higher.
How do I convert a molar ratio to mass?
insert mass = ratio × vector mass × (insert length / vector length). Enter your values above to compute it.
Why not just use equal masses of insert and vector?
Ligation depends on molecule number. Equal masses give very different molar amounts when the fragments differ in length.
Related guide: Restriction enzyme cloning: a step-by-step guide →
