About the Ligation Calculator
This ligation calculator tells you how much insert DNA to add to a ligation reaction to hit a chosen insert:vector molar ratio. Enter the amount of linearised vector you plan to use, the lengths of vector and insert in base pairs, and the ratio you want — 3:1 is the usual starting point for a single-insert cloning reaction. The calculator returns the insert mass in nanograms, the femtomoles of each fragment, and, if you give the stock concentrations, the volumes to pipette.
It is meant for molecular biology bench work: restriction cloning, sticky- and blunt-end ligations and subcloning PCR products. Because molar ratios depend on fragment length, a short insert needs far fewer nanograms than a long one to reach the same number of molecules, and getting this wrong is a common reason for empty-vector colonies.
Lengths are double-stranded base pairs. Picomole values use the average molecular weight of dsDNA (617.96 g/mol per base pair plus 36.04 g/mol for the ends), and volumes assume the stock concentrations you enter are accurate — quantify DNA after gel purification for best results.
With the default inputs, the insert mass needed is 33.33 ng. Change any value above to recalculate instantly.
How to use the ligation calculator
- 1Enter how many nanograms of cut vector you will use.
- 2Enter the vector and insert lengths in base pairs.
- 3Set the insert:vector molar ratio (3 for 3:1).
- 4Optionally add the stock concentrations to get pipetting volumes.
- 5Read the insert mass and compare other ratios in the table.
Formula and method
A molar ratio compares numbers of DNA molecules, not masses. Because a molecule’s mass is proportional to its length, the mass of insert that contains the same number of molecules as your vector is the vector mass scaled by insert length ÷ vector length. Multiplying by the desired ratio gives the insert mass.
The molecular weight of double-stranded DNA is approximately 617.96 g/mol per base pair plus 36.04 g/mol for the two ends, so moles = mass ÷ MW. The calculator reports femtomoles because a typical ligation contains 10–100 fmol of vector ends. Volumes are mass ÷ stock concentration.
- ng
- DNA mass in nanograms
- bp
- Fragment length in base pairs
- ratio
- Desired insert:vector molar ratio (e.g. 3 for 3:1)
- MW
- dsDNA molecular weight ≈ 617.96 × bp + 36.04 g/mol
Worked examples
1.2 kb insert into a 5.4 kb vector at 3:1
Insert mass = 50 ng × (1200 ÷ 5400) × 3 = 33.3 ng. At 20 ng/µL that is 1.67 µL of insert with 1.0 µL of vector. The vector is about 15 fmol and the insert about 45 fmol — a true 3:1 molar ratio.
500 bp insert into a 3 kb vector at 1:1
With equal molecule numbers, the insert mass is just 100 ng × 500 ÷ 3000 = 16.7 ng — one-sixth of the vector mass because it is one-sixth as long.
Short 300 bp fragment at 5:1
Short inserts are often ligated at a higher ratio. Insert mass = 25 × (300 ÷ 6000) × 5 = 6.25 ng, so only a few nanograms are needed even at a 5-fold molar excess.
Frequently asked questions
What insert to vector ratio should I use for ligation?+
A 3:1 insert:vector molar ratio is the standard starting point for single-insert cloning. Use 1:1 to 3:1 for inserts close to the vector size and 5:1 to 7:1 for short inserts; test a few ratios if colonies are scarce.
Why can’t I just use a 3:1 mass ratio?+
Because ligation depends on the number of DNA ends meeting, and a long fragment has fewer molecules per nanogram than a short one. Mass ratios only equal molar ratios when the insert and vector are the same length.
How much vector should I use in a ligation?+
Most protocols use 20–100 ng of dephosphorylated or double-cut vector in a 10–20 µL reaction, which is roughly 10–50 fmol for a typical 3–6 kb plasmid backbone.
What molecular weight is used for DNA?+
The calculator uses 617.96 g/mol per base pair plus 36.04 g/mol, the average for double-stranded DNA with typical base composition. The older rule of thumb of 650 g/mol per bp gives values within about 5%.
Does this work for Gibson or In-Fusion assembly?+
The same molar arithmetic applies, though those methods usually recommend 2:1 to 3:1 insert:vector and specific total amounts in pmol. Check the kit instructions for the recommended ratio and enter it here.