Plasmid Map
Tools

Ligation calculator

Turn an insert:vector molar ratio into DNA masses and pipetting volumes.

01

Your values

bp
bp
ng
: 1

Pipetting volumes Optional

Enter the measured DNA concentrations to convert masses to volumes.

ng/µL
ng/µL

Calculations stay in this browser. No account or upload.

02

Example result

Insert DNA required

50ng

50 ng of insert with 50 ng of vector gives an insert:vector molar ratio of 3:1.

Vector volume
2.5 µL
Insert volume
5 µL
Calculation & assumptions

50 ng × (1000 bp ÷ 3000 bp) × 3 = 50 ng

  • For double-stranded DNA, using length as proportional to molecular mass. Ratios compare molecules, not masses.
  • DNA volumes exclude buffer, ligase and water. The chosen ratio is not a prediction of ligation yield.

A molar ratio counts DNA molecules

An insert:vector molar ratio compares the numbers of molecules. It is not generally the same as the ratio of their masses, because an insert and a vector can have different lengths. For double-stranded DNA, using length as proportional to molecular mass gives:

Insert mass = vector mass × (insert length / vector length) × insert:vector molar ratio.

Enter both lengths in base pairs, the vector mass in nanograms and the desired ratio. For a 3:1 insert:vector molar ratio, enter 3. The ratio is an input to this calculation, not a recommendation automatically selected for your experiment.

Worked example: a 1 kb insert and a 3 kb vector

Take 50 ng of a 3,000 bp vector and a 1,000 bp insert. At a 3:1 insert:vector molar ratio, the required insert mass is 50 × (1,000 / 3,000) × 3 = 50 ng. The masses happen to be equal here because the vector is three times as long as the insert.

Changing the ratio to 1:1 gives approximately 16.667 ng of insert. Increasing the insert length while holding the molar ratio fixed increases the required insert mass. These relationships are useful checks when comparing a calculation with an experimental plan.

Convert the DNA masses to volumes

Enter your measured vector and insert concentrations in ng/µL to calculate the volume of each DNA preparation. In the example, a 20 ng/µL vector stock supplies 50 ng in 2.5 µL. A 10 ng/µL insert stock supplies 50 ng in 5 µL. Each optional concentration is handled independently; leave a field empty if you only need the mass.

These volumes cover the DNA preparations only. They do not include ligase, buffer, water or a final reaction-volume constraint. The calculation does not assess end compatibility, phosphorylation, purity, ligation yield or transformation efficiency.

Use the lengths of the fragments you will combine

Open your annotated file in the sequence editor to inspect the map and coordinates. Use the length of the intended vector backbone after preparation, rather than automatically using the length of an uncut source plasmid. Joined feature segments and excised intervals can have different lengths from the whole record.

The mass-ratio relationship is also documented by NEB's ligation calculator. This tool uses its own local implementation. Copy or save the calculation to keep the selected ratio, lengths, masses and optional concentration settings together.