Ligation Protocol for Cloning with ElectroLigase® (M0369)
- Transfer ElectroLigase® and ElectroLigase®
Reaction Buffer to ice prior to reaction set up. Mix tubes by finger flicking
- 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 ElectroLigase Reaction Buffer
and 1 μl of ElectroLigase and pipet up and down 7–10 times to
- Incubate ligation reaction at room
temperature (25°C) for 30–60 minutes.
- Inactivate the ligase by incubating the
reaction at 65°C for 15 minutes.
- Chill sample on ice (if to be used within
a few hours) or store at -20°C.
* 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.