Ligation calculator: insert mass for any insert:vector molar ratio
For a ligation, ng of insert = molar ratio × ng of vector × (insert length in bp / vector length in bp). With 50 ng of a 5,400 bp vector and a 1,200 bp insert, a 3:1 insert:vector ratio needs 33.3 ng of insert; the calculator also gives the pmol of each piece and the amounts for multi-fragment assemblies.
Last updated
Loading Ligation calculator…
At a glance
| Formula | ng insert = ratio × ng vector × insert bp / vector bp |
|---|---|
| Moles of DNA | pmol = ng × 1000 / (bp × 650) |
| Ratios shown | 1:1, 2:1, 3:1, 4:1 and your own |
| Assembly mode | Any number of fragments, each at its own molar ratio |
| Output | ng, pmol and pipetting volumes; CSV |
| Runs in | Your web browser; nothing is uploaded |
| Price | Free, no account needed |
What it does
How to use the ligation calculator
- Enter the vectorType the length of the linearised vector in bp and the mass you will use in ng.
- Enter the insertType the insert length in bp and pick an insert:vector molar ratio, such as 3 for 3:1.
- Add stock concentrationsOptional: enter each stock in ng/µL to get the volume to pipette.
- For an assembly, add fragmentsSwitch to Multi-fragment assembly and list each fragment with its length and molar ratio to the vector.
- Read and copyRead the ng and pmol of each piece, then copy the result or download the CSV.
How it works
pmol = ng × 1000 / (bp × 650)
ng insert = ratio × ng vector × insert bp / vector bp
A ligation ratio is a ratio of molecules (moles), not of masses. The moles of double-stranded DNA are its mass divided by its molar mass, and its molar mass is the length in base pairs times an average of 650 g/mol per base pair, the value used for plasmid copy numbers by Na et al. (2026). So a fragment's pmol is ng × 1000 / (bp × 650). Because every base pair has the same average mass, equal moles of a longer fragment weigh proportionally more, and the insert mass for a molar ratio is the vector mass times the ratio times the length ratio.
The ratio matters: Green and Sambrook (2020) explain that too much vector relative to insert gives many empty circular plasmids, and too little gives chains of several inserts and vectors, so the number and orientation of inserts should be checked in the clones. Speer and Richard (2011) compared insert:vector ratios of 1:1, 2:1 and 4:1 in their sticky-end assembly, and 4:1 gave the highest accuracy in three of the four assemblies they tested. Use the ratio your ligase or kit protocol recommends.
For multi-fragment assemblies each fragment is given its own molar ratio to the vector. Kits specify their own total DNA; for example, the GeneArt Gibson HiFi protocol of Cisneros et al. (2025) uses 50–100 ng of linearised vector, a 5:1 insert:vector ratio for an insert shorter than 200 bp, and 0.02–0.5 pmol of DNA in total.
Sources: Na Q et al. TaqMan qPCR quantification of the Cytb gene for precise determination of camel milk in dairy products. PLoS One 2026;21:e0342999, doi:10.1371/journal.pone.0342999. Speer MA, Richard TL. Amplified insert assembly: an optimized approach to standard assembly of BioBrick genetic circuits. J Biol Eng 2011;5:17, doi:10.1186/1754-1611-5-17. Green MR, Sambrook J. Cloning in plasmid vectors: blunt-end cloning. Cold Spring Harb Protoc 2020, doi:10.1101/pdb.prot101246. Cisneros AE et al. Effective gene silencing in plants by synthetic trans-acting siRNAs derived from minimal precursors. Bio-protocol 2025;15:e5475, doi:10.21769/BioProtoc.5475.
Worked example
Ligation. 50 ng of a 5,400 bp linearised vector is 50 × 1000 / (5,400 × 650) = 0.0142 pmol. For a 1,200 bp insert at 3:1, the insert is 3 × 0.0142 = 0.0427 pmol, which is 3 × 50 ng × 1,200 / 5,400 = 33.3 ng. At 1:1, 2:1 and 4:1 the insert would be 11.1, 22.2 and 44.4 ng.
Assembly. With the same 50 ng of vector and two fragments of 1,500 bp and 800 bp, each at 2:1 to the vector (0.0285 pmol each), you need 27.8 ng of the 1,500 bp fragment and 14.8 ng of the 800 bp fragment: 0.0712 pmol and 92.6 ng of DNA in all.
Frequently asked questions
How do I calculate how much insert to use in a ligation?
Multiply the mass of vector by the insert:vector molar ratio and by the insert length divided by the vector length: ng insert = ratio × ng vector × insert bp / vector bp. For 50 ng of a 5,400 bp vector and a 1,200 bp insert at 3:1, that is 33.3 ng.
What insert to vector ratio should I use?
Use the ratio your ligase or kit protocol gives. Speer and Richard (2011) compared 1:1, 2:1 and 4:1 (insert:vector) and found 4:1 most accurate in three of four assemblies; one Gibson HiFi protocol uses 5:1 for inserts shorter than 200 bp (Cisneros et al. 2025).
Why is the ratio molar and not by mass?
Ligation joins molecules end to end, so what matters is the number of insert ends per vector end. A short insert has many more molecules per nanogram than a long vector, so equal masses would give a large molar excess of insert.
How do I convert ng of DNA to pmol?
Divide the mass by the molar mass: pmol = ng × 1000 / (length in bp × 650), using an average of 650 g/mol per base pair of double-stranded DNA (Na et al. 2026). 50 ng of a 5,400 bp plasmid is 0.0142 pmol.
What goes wrong if the ratio is off?
Too much vector gives many empty re-circularised plasmids; too little vector gives chains of several inserts and vectors (Green and Sambrook 2020). Check the number and orientation of inserts in your clones by restriction mapping or sequencing.
Should I use the length of the whole plasmid or the cut vector?
Use the length of the vector as it goes into the reaction, after cutting or linearising, because that is the molecule whose moles you are counting.
Does this work for Gibson or HiFi assembly?
Yes: switch to Multi-fragment assembly, give each fragment its molar ratio to the vector, and check the total DNA against your kit’s manual.
Make the whole figure in LabCanvas
Vector figure editor, slides, 3D models, protein structures and Prism-style graphs, in your browser.
Get started