Ligation Protocol with T4 DNA Ligase (M0202) also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 


  1. Set up the following reaction in a microcentrifuge tube on ice.
    (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.
    T4 DNA Ligase Buffer (10X)* 2 μl
    Vector DNA (4 kb) 50 ng (0.020 pmol)
    Insert DNA (1 kb) 37.5 ng (0.060 pmol)
    Nuclease-free water to 20 μl
    T4 DNA Ligase 1 μl
    * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
  2. Gently mix the reaction by pipetting up and down and microfuge briefly.
  3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
  4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
  5. Heat inactivate at 65°C for 10 minutes.
  6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
  1. NEBioCalculator Ligations Thumbnail

    NEBioCalculator® - Using the Ligation module

    This tutorial describes the use of the NEBioCalculator web tool to optimize the molar ratio between vector and insert DNA for use in a ligation reaction.