About this calculator
The Ligation Calculator works out how much insert DNA to combine with a given amount of vector DNA for a cloning reaction, based on their sizes and your target molar ratio.
How is the insert amount calculated?
The calculation converts vector mass into moles (using the vector's length in base pairs), applies the desired insert-to-vector molar ratio, and converts that molar amount of insert back into a mass using the insert's own length. In formula form: Insert Mass = Vector Mass × (Insert Length ÷ Vector Length) × Molar Ratio (Insert:Vector).
Why does molar ratio matter more than mass ratio?
DNA ligation reactions depend on how many insert and vector molecules are available to find and join each other — not on their total mass. Because insert fragments are usually much shorter than the vector, using a mass ratio instead of a molar ratio would badly under- or over-represent the actual number of insert molecules present, reducing ligation efficiency.
What molar ratio should you use?
A commonly used starting point is a 3:1 insert-to-vector molar ratio, though optimal ratios vary depending on insert size, ligase efficiency, and the specific cloning method — it's common to set up several reactions across a range of ratios (for example 1:1, 3:1, and 5:1) and see which produces the most successful transformants.