Why use a molar ratio
Ligation depends on the number of DNA ends, not their mass. A small insert has many more molecules per ng than a large vector, so equal masses would give very unequal numbers of molecules. Protocols therefore give a molar ratio of insert to vector, most often 3:1.
The formula
insert (ng) = vector (ng) × insert length ÷ vector length × ratio
With 50 ng of a 3,000 bp vector and a 1,000 bp insert at 3:1: 50 × 1,000 ÷ 3,000 × 3 = 50 ng of insert.
Tips
- Try 1:1, 3:1 and 5:1 if the first attempt gives few colonies.
- Very short inserts, such as annealed oligos, often need higher ratios.
- Include a vector-only ligation as a control for background from uncut or self-ligated vector.
- Measure concentrations after purification, not before.
Calculator
The Ligation Calculator gives the ng of insert for any ratio and, with the insert concentration, the volume to pipette. Measure that concentration with the DNA Concentration Calculator.