FAQ: How much DNA should be used in a ligation using T3 DNA Ligase?

To promote circle formation, useful in transformation, the overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector molar ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.
  1. NEBTV_DNA-Modifying-Enzymes-and-Cloning-Techniques_1920

    Quick Tips - How do I calculate how much DNA to add to a ligation reaction?

    This video provides tips for calculating the ideal insert:vector molar ratio before performing a ligation reaction.