Ligation Protocol with T3 DNA Ligase (M0317)

  1. Set up the following reaction in a microcentrifuge tube on ice.
    (T3 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.
    StickTogether DNA Ligase Buffer (2X) 10 μl
    Vector DNA (4 kb) 50 ng (0.020 pmol)
    Insert DNA (1 kb) 37.5 ng (0.060 pmol)
    T3 DNA Ligase 1 μl
    Nuclease-free water to 20 μl
    * The T3 DNA Ligase Reaction Buffer should be thawed and resuspended at room temperature.

  2. Gently mix the reaction by pipetting up and down and microfuge briefly.
  3. Incubate at room temperature (25°C) for 15-30 minutes.
  4. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Alternatively, store at –20°C.
  5. Do not heat inactivate – Heat inactivation dramatically reduces transformation efficiency.
  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.