DNA Ligation Calculator — Insert Mass & Molar Ratio
Determine exactly how many nanograms of insert DNA to add to your ligation: enter the vector mass, vector and insert sizes in kilobases, and the desired molar ratio. The calculator also reports the pmol of each fragment.
ng
kb
kb
Mass of insert DNA to add to the ligation reaction
- 1
Size ratio = kb insert ÷ kb vector
1 ÷ 3 = 0.3333Converts molar ratio to a mass ratio, since larger fragments weigh more per molecule. - 2
Base insert mass = ng vector × size ratio
100 × 0.3333 = 33.33 - 3
Insert mass = base × molar ratio
33.33 × 3 = 100
How does this calculator work?
Use ng insert = (ng vector × kb insert / kb vector) × molar ratio. A 100 ng, 3 kb vector with a 1 kb insert at 3:1 molar ratio needs ≈ 33 ng of insert. The 660 Da/bp constant converts mass to moles for each fragment. Standard molar ratio is 3:1 for sticky ends.
Formula
How this is calculated
A ligation reaction joins a linearised vector to an insert fragment using DNA ligase. The efficiency of ligation depends on the molar ratio of insert to vector, not the mass ratio — because larger fragments weigh more per molecule. The formula ng insert = (ng vector × kb insert / kb vector) × molar ratio converts a chosen molar ratio into a mass you can pipette.
The conversion between mass and moles uses the average molecular weight of double-stranded DNA, approximately 660 g/mol per base pair (660 Da/bp). So pmol = ng / (kb × 660). A 3 kb vector at 100 ng is roughly 0.0505 pmol; adding insert at a 3:1 molar ratio requires ≈ 0.1515 pmol, whose mass you get from the formula above.
Recommended ratios vary by cloning strategy: sticky-end ligations typically use 3:1 (insert:vector), blunt-end ligations sometimes 5–10:1 because efficiency is lower. Total DNA in the reaction is generally kept below 200–300 ng; if the calculated insert mass pushes the total above that, scale down the vector mass proportionally. These values are estimates — your lab protocol, vector topology, and ligase brand may call for optimisation.
Frequently asked questions
For sticky-end ligations a 3:1 molar ratio (insert:vector) is standard. For blunt-end or T-A ligations where efficiency is lower, 5:1 or even 10:1 is often used. Some labs optimise by running a dilution series of ratios on the same day.
DNA ligase acts on molecular ends, so it is the number of molecules — not their mass — that determines whether ends find each other. A 3 kb insert and a 3 kb vector contribute equal ends per molecule; a 1 kb insert contributes the same number of ends but weighs three times less, so you must add three times more mass to achieve the same molar ratio.
Double-stranded DNA has an average molecular weight of about 660 daltons (g/mol) per base pair, accounting for both strands and the average nucleotide composition. For single-stranded DNA the constant is roughly 330 Da/nt. This lets you convert between nanograms and picomoles when you know the fragment size in kilobases.
Also known as
TG we-Calculate Editorial Team. (2026). DNA Ligation Calculator — Insert Mass & Molar Ratio [Online calculator]. TG we-Calculate. https://we-calculate.com/calculator/ligation-calculator
TG we-Calculate Editorial Team. "DNA Ligation Calculator — Insert Mass & Molar Ratio." TG we-Calculate. 2026. https://we-calculate.com/calculator/ligation-calculator.
TG we-Calculate Editorial Team, "DNA Ligation Calculator — Insert Mass & Molar Ratio," TG we-Calculate, 2026. [Online]. Available: https://we-calculate.com/calculator/ligation-calculator
@misc{wecalculate_ligation_calculator, title = {DNA Ligation Calculator — Insert Mass & Molar Ratio}, author = {{TG we-Calculate Editorial Team}}, howpublished = {\url{https://we-calculate.com/calculator/ligation-calculator}}, year = {2026}, note = {TG we-Calculate} }
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