FAQ: How much DNA should be used in a ligation using T4 DNA Ligase?

The unit definition uses 0.12 μM (300 μg/ml) lambda HindIII fragments. The high DNA concentration can be used for linker ligation. To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Vector:Insert molar ratios between 1:1 and 1:10 are recommended (1:3 is typical). If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.