• My NEB
  • Print
  • PDF
  • Ligation Protocol for Cloning with Blunt/TA Ligase Master Mix (M0367)

    Protocol

    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.