Ligation Insert:Vector Ratio Calculator
Work out how many nanograms of insert to add for a chosen insert:vector molar ratio from the fragment sizes and vector mass.
How it works
Insert (ng) = vector (ng) × (insert bp ÷ vector bp) × molar ratio · fmol ≈ ng ÷ (bp × 0.00065)
Ligation efficiency depends on molecules, not mass: a short insert weighs less per molecule, so equal nanograms would swamp the vector. Scaling by the length ratio equalises moles, and a 3:1 insert excess is the usual starting point for sticky ends (1:1 to 5:1 range; blunt ends benefit from higher). Too much insert promotes concatemers.
Worked example
50 ng of a 3,000 bp vector, 750 bp insert at 3:1: 50 × 0.25 × 3 = 37.5 ng of insert.
Frequently asked questions
How much vector should I use?
25–100 ng in a 10–20 µL reaction. Gibson and Golden Gate assemblies use their own molar rules (often 2:1 with 50–100 fmol total).
Where does 0.00065 come from?
One base pair averages 650 g/mol, so 1 fmol of a 1 bp fragment is 0.00065 ng.
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For teaching and planning — verify against your lab protocol and instrument documentation.