Ligation calculator.
Set up a DNA ligation. Enter your vector mass and length, the insert length and the molar ratio you want, and the calculator gives the mass of insert to add.
Vector and insert
LiveInsert mass = vector mass x (insert length / vector length) x molar ratio. A 3:1 insert to vector ratio suits most sticky-end ligations.
Insert to use
30 ng
of insert for this ligation
A planning estimate; optimal ratios vary by ends and enzyme. Calculations run in your browser; nothing you enter is stored.
How it works
Matching the molar ratio
A ligation joins an insert into a vector, and efficiency depends on their molar ratio, not their mass. Because longer DNA weighs more per molecule, you scale the insert mass by the ratio of insert to vector length. The insert mass is the vector mass, times insert length over vector length, times the molar ratio.
With 50 ng of a 5,000 bp vector and a 1,000 bp insert at a 3:1 molar ratio, you add 30 ng of insert.
Reference
Molar ratio to insert mass.
Insert mass for a 50 ng, 5,000 bp vector with a 1,000 bp insert at different ratios.
| Molar ratio | Insert mass |
|---|---|
| 1:1 | 10 ng |
| 2:1 | 20 ng |
| 3:1 | 30 ng |
| 5:1 | 50 ng |
| 7:1 | 70 ng |
The full guide
The complete guide to ligation ratios.
Why molar ratio matters, how to calculate the insert mass, and which ratios to use.
Why molar ratio, not mass
Ligation depends on the number of insert and vector molecules meeting, so it is the molar ratio that matters. Equal masses of a long vector and a short insert hold very different numbers of molecules, which is why you cannot just match masses.
Scaling by length corrects for this: a shorter insert needs less mass to reach the same number of molecules as the vector.
Calculating the insert mass
Take the vector mass, multiply by the insert length divided by the vector length, then multiply by the molar ratio you want. The result is the insert mass to add. The calculator does this instantly as you change any value.
Keep lengths in the same units, base pairs, and the masses come out in the same unit as the vector mass you enter.
Which ratio to use
A 3:1 insert to vector molar ratio is the usual starting point for sticky-end ligations. Blunt-end ligations are less efficient, so higher ratios such as 5:1 often work better. If you get few colonies, try a range of ratios.
Too little insert favours empty vector re-circularising; too much can cause multiple inserts, so the ratio is a balance.
Running the ligation
Use enough total DNA to be pipettable but not so much that volumes get awkward, typically tens to low hundreds of nanograms of vector. Include a vector-only control to gauge background from self-ligation.
Once you have the insert mass from the calculator, set up the reaction with buffer and ligase per your kit.
The formula
Molecules,
not mass.
Insert mass is vector mass times the length ratio times the molar ratio. 3:1 suits most sticky ends.
DNA copy number ›# Insert mass for a ligation
insert_ng = vector_ng × (insert_bp / vector_bp) × ratio
# worked example
50 × (1000/5000) × 3 = 30 ngQuestions
Ligation questions.
How do I calculate insert amount for a ligation?
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Insert mass equals the vector mass times the insert length over the vector length, times the molar ratio. With 50 ng of a 5,000 bp vector, a 1,000 bp insert at 3:1 needs 30 ng.
What insert to vector ratio should I use?
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A 3:1 insert to vector molar ratio is the standard for sticky-end ligations. Blunt ends often work better at 5:1 or higher. Try a range if colonies are scarce.
Why scale by length?
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Because ligation depends on the number of molecules, not mass. A longer fragment weighs more per molecule, so the length ratio converts a molar ratio into the right mass.
How much vector should I use?
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Typically tens to low hundreds of nanograms, enough to pipette accurately. Always run a vector-only control to check for self-ligation background.
Is this ligation calculator free?
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Yes. It is completely free with no sign-up, and every calculation runs locally in your browser, so nothing you enter is stored or sent anywhere.
About the developer
Jean Borg
Jean builds and maintains every calculator on freecalculators.pro from Malta, with a focus on tools that are fast, free and show their working. The ligation calculator uses the standard insert-to-vector molar ratio formula and is for lab planning.