What ratio should I use?

A 3:1 insert:vector molar ratio is a common default for cohesive-end ligations; blunt-end ligations sometimes use higher ratios. Optimise empirically.

Why does length appear in the formula?

Molar ratio is about numbers of molecules. For the same mass, a shorter fragment contains more molecules, so the mass needed scales with the insert-to-vector length ratio.

Ligation molar ratio

Compute the insert mass needed for a target insert:vector molar ratio in a ligation, from vector mass and the two fragment lengths.

How it works

Formula

insert mass = vector mass × (insert length ÷ vector length) × molar ratio. Mass scales with length because a longer fragment has more mass per mole.

Worked example

50 ng of a 5,000 bp vector with a 1,000 bp insert at a 3:1 insert:vector ratio: 50 × (1,000 ÷ 5,000) × 3 = 30 ng of insert.

When to use it

When setting up a ligation or Gibson/assembly reaction and you need the insert mass that gives a chosen molar ratio to a known amount of vector.

Sensible defaults

Defaults use 50 ng of a 5 kb vector, a 1 kb insert and a 3:1 ratio — a common cohesive-end ligation starting point. Try 1:1 to 5:1 to optimise.

FAQ

What ratio should I use?: A 3:1 insert:vector molar ratio is a common default for cohesive-end ligations; blunt-end ligations sometimes use higher ratios. Optimise empirically.
Why does length appear in the formula?: Molar ratio is about numbers of molecules. For the same mass, a shorter fragment contains more molecules, so the mass needed scales with the insert-to-vector length ratio.