Ligation Protocol for Cloning with Blunt/TA Ligase Master Mix (M0367)
- Transfer master mix to ice prior to reaction set up. Mix tube by finger flicking before use.
- Combine 20–100 ng of vector* with a 3-fold molar excess of insert and adjust volume to 5 μl with dH2O.
- Add 5 μl of Blunt/TA Ligase Master Mix and mix thoroughly by pipetting up and down 7-10 times or by finger-flicking.
- Incubate at room temperature (25°C) for 15 min, place on ice.
- Use for transformation or store at -20°C.
- 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.
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.