Ligation Protocol for Cloning with ElectroLigase® (M0369)

Protocol

  1. Transfer ElectroLigase® and ElectroLigase® Reaction Buffer to ice prior to reaction set up. Mix tubes 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 ElectroLigase Reaction Buffer and 1 μl of ElectroLigase and pipet up and down 7–10 times to mix.

  4. Incubate ligation reaction at room temperature (25°C) for 30–60 minutes.

  5. Inactivate the ligase by incubating the reaction at 65°C for 15 minutes.

  6. 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.