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  • 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.