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.
A worked example with volumes
From the example above you need 50 ng of vector and 50 ng of insert. If the vector is at 50 ng/µL and the insert at 25 ng/µL:
- Vector: 50 ÷ 50 = 1 µL.
- Insert: 50 ÷ 25 = 2 µL.
- 10× ligase buffer: 2 µL. T4 DNA ligase: 1 µL.
- Water to 20 µL: 14 µL.
Checking it in moles
A base pair of double-stranded DNA weighs about 650 g/mol, so pmol = ng × 1,000 ÷ (bp × 650). The vector is 50,000 ÷ (3,000 × 650) ≈ 0.026 pmol and the insert 50,000 ÷ (1,000 × 650) ≈ 0.077 pmol, three times as much.
Few or no colonies
- The ATP in ligase buffer breaks down with repeated freezing and thawing. Keep the buffer in small aliquots.
- Blunt ends ligate far less readily than sticky ends; use more ligase and a longer incubation.
- A dephosphorylated vector needs an insert that still has its 5′ phosphates; PCR products made with ordinary primers do not.