Ligation Protocol for Cloning with Instant Sticky-end Ligase Master Mix (M0370)
- 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
- 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.
- Use for transformation or store at
- Do not heat
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.
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.