Ligation calculator (insert : vector)
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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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
- What is a good insert:vector ratio for sticky-end ligation?
- 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.
- Do vector and insert lengths need to be in kb?
- 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.
- Does this work for Gibson or Golden Gate assembly?
- 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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