Ligation calculator (insert : vector)
Maintained by the Talindrew team · Last updated
The insert mass for a ligation is the vector mass scaled by the length ratio and the desired molar ratio: ng insert = ng vector × (insert length / vector length) × (insert:vector ratio). For 100 ng of a 3 kb vector with a 0.5 kb insert at 3:1, add 50 ng of insert.
Result
ng insert = ng vector × (insert kb / vector kb) × ratio. A 3:1 insert:vector molar ratio is the usual starting point for sticky-end ligations.
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What it looks like
Formula
ng insert = ng vector × (insert kb / vector kb) × (insert:vector molar ratio)
Worked example
Given: 100 ng of 3 kb vector, 0.5 kb insert, 3:1 insert:vector
- 1.Length ratio: 0.5 / 3 = 0.1667.
- 2.Equimolar insert mass: 100 ng × 0.1667 = 16.7 ng.
- 3.At 3:1 molar excess: 16.7 × 3 = 50 ng.
Add 50 ng of insert
How the calculation flows
Units & constants
| Typical ratio, sticky ends | 3:1 insert:vector (try 1:1 to 5:1) |
|---|---|
| Typical ratio, blunt ends | 1:1 to 5:1 with higher total DNA |
| Typical vector amount | 50–100 ng per 10–20 µl reaction |
| Basis | Molar ratio: mass is scaled by fragment length |
| Price | Free |
Why is the ratio molar rather than by mass?
Ligation joins molecules one-to-one, so what matters is the count of insert molecules per vector molecule. A 0.5 kb insert weighs six times less per molecule than a 3 kb vector; mixing equal masses would actually be a 6:1 molar excess of insert. The length ratio in the formula is exactly the correction that converts a mass ratio into a molar ratio.
What ratio should you use?
3:1 insert:vector is the standard starting point for cohesive-end ligations.
- Religating background high (empty vector colonies)? Increase insert excess to 5:1, or dephosphorylate the vector.
- Insert concatemers or multiple inserts? Drop toward 1:1.
- Very small inserts (< 100 bp, linkers): ratios of 10:1 or more are common.
- Large inserts approaching vector size: 1:1 to 2:1 works better than a large excess.
How much total DNA belongs in a ligation?
For a typical 10–20 µl T4 DNA ligase reaction, 50–100 ng of vector plus the calculated insert keeps total DNA in the 1–10 ng/µl range where intermolecular joining is favoured. Very high DNA concentrations push toward concatemers; very low ones favour self-circularisation of the vector.
Frequently asked questions
Start at 3:1. If you see high empty-vector background, raise the insert excess to 5:1 or dephosphorylate the vector; if you see multiple inserts, drop to 1:1. The optimum is construct-specific, which is why the calculator lets you set any ratio.
They need to be in the same unit, both kb or both bp. Only the ratio of the two lengths enters the formula, so the unit cancels.
The same molar arithmetic applies: those protocols typically call for equimolar fragments (1:1) or a 2:1 insert excess, with total amounts specified in pmol. Use the dsDNA mass↔moles converter to get each fragment's pmol from its mass.
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