Insert (ng) = Vector (ng) × (Insert size / Vector size) × Ratio
Ligation joins a DNA insert into a linearized vector using T4 DNA ligase. The key to successful ligation is the molar ratio of insert to vector. Formula: insert (ng) = vector (ng) × (insert size / vector size) × molar ratio. A 3:1 insert:vector ratio is most common. For sticky-end ligations: 16°C overnight or 25°C for 10 minutes (quick ligase). For blunt-end ligations: higher insert ratios (5:1-10:1) and more ligase are needed. Total DNA should be 50-100 ng in a 10-20 µL reaction. Include controls: vector only (tests for self-ligation), no ligase (tests for uncut vector). After ligation, transform into competent E. coli cells.