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.