Home Protocols Ligation Protocol for Cloning with Instant Sticky-end Ligase Master Mix (M0370) 

Ligation Protocol for Cloning with Instant Sticky-end Ligase Master Mix (M0370) 

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 Instant Sticky-end Ligase Master Mix, mix thoroughly by pipetting up and down 7-10 times, and place on ice. The sample is now ready to be used for transformation.
  4. Use for transformation or store at -20°C.
  5. 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 (sticky) and a corresponding 3-fold molar excess of the insert to be ligated into the vector.

Videos

  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.