Ligation Protocol for Cloning with Blunt/TA Ligase Master Mix (M0367)


  1. Transfer master mix to ice prior to reaction set up. Mix tube by finger flicking before use.

  2. Combine 20–100 ng of vector* with a 3-fold molar excess of insert and adjust volume to 5 μl with dH2O.

  3. Add 5 μl of Blunt/TA Ligase Master Mix and mix thoroughly by pipetting up and down 7-10 times or by finger-flicking.

  4. Incubate at room temperature (25°C) for 15 min, place on ice.

  5. Use for transformation or store at -20°C.

  6. Do not heat inactivate.

    Heat inactivation dramatically reduces transformation efficiency.

    * In-house testing has demonstrated that maximal transformation efficiency is achieved using between 20–100 ng of vector (blunt or sticky, including T-vectors) and a corresponding 3-fold molar excess of the insert to be ligated into the vector.
  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.