Protocol for NEBridge™ Ligase Master Mix (NEB #M1100)

  1. Based on assembly complexity, determine reaction component volumes (Table 1). The volume of Type IIS restriction enzyme (x µl) can be found in Table 2.

    Table 1: Assembly Component Amounts

    COMPONENTS 2 FRAGMENT OR
    3–6 FRAGMENT ASSEMBLY
    7+ FRAGMENT
    NEBridge Ligase Master Mix 5 µl 10 µl
    DNA Fragments* 0.05 pmol each 0.05 pmol each
    Type IIS Restriction Enzyme x µl x µl
    Nuclease-free Water y µl y µl
    Total Reaction Volume 15 µl 30 µl

    * Use NEBiocalculator® to calculate the mass of each DNA fragment

    Table 2: Suggested Type IIS Restriction Enzyme Amounts

    ENZYME 2 FRAGMENT ASSEMBLY 3–6 FRAGMENT ASSEMBLY >7+ FRAGMENT ASSEMBLY
    BbsI-HF (NEB #R3539) 1 µl (20 U) 1 µl (20 U) 1 µl (50 U)*
    BsaI-HFv2 (NEB #R3733) 1 µl (20 U) 1 µl (20 U) 1 µl (20 U)
    BsmBI-v2 (NEB #R0739) 3 µl (30 U) 3 µl (30 U) 6 µl (60 U)
    Esp3I (NEB #R0734) 2 µl (20 U) 3 µl (30 U) 4 µl (40 U)
    PaqCI (NEB #R0745)** 1 µl (10 U) 1 µl (10 U) 2.5 µl (25 U)
    SapI (NEB #R0569) 1 µl (10 U) 1 µl (10 U) 2 µl (10 U)
    BspQI (NEB #R0712) 1 µl (10 U) 1 µl (10 U) 2 µl (10 U)

    * Use BbsI-HF (NEB #R3539M) (50 U/µl).
    ** Requires PaqCI activator (20 µM), 0.5 µl for 2 and 3–6 fragment assembly; 1.25 µl for 7+ fragment assembly.

  2. Set up a reaction in a microcentrifuge tube on ice. Mix DNA fragments (0.05 pmol of each) with nuclease-free water (y µl).

  3. Add NEBridge Ligase Master Mix (5 µl or 10 µl) to DNA fragments and water. Gently mix by pipetting 3 times.

  4. Add Type IIS restriction enzyme (x µl). Gently mix by pipetting 5 times.

  5. Incubate for the recommended time and temperature (see Table 3).

  6. Table 3: Suggested cycle times 

      2 FRAGMENT ASSEMBLY
      SINGLE GENE CLONING LIBRARY CONSTRUCTION
    BbsI-HF, BsaI-HFv2, Esp3I, PaqCI, SapI 37ºC for 15 min. 37ºC for 60 min.
    BsmBI-v2, BspQI  15 cycles at 42ºC for 1 min. and 16ºC for 1 min. 30 cycles at 42ºC for 1 min. and 16ºC for 1 min.


      3–6 FRAGMENT ASSEMBLY 7+ FRAGMENT ASSEMBLY
    7–13 FRAGMENT 14+ FRAGMENT
    BbsI-HF, BsaI-HFv2, Esp3I, PaqCI, SapI 30 cycles at 37ºC for 1 min. and 16ºC for 1 min. 30 cycles at 37ºC for 5 min. and 16ºC for 5 min. 60 cycles at 37ºC for 5 min. and 16ºC for 5 min.
    BsmBI-v2, BspQI 30 cycles at 42ºC for 1 min. and 16ºC for 1 min. 30 cycles at 42ºC for 5 min. and 16ºC for 5 min. 60 cycles at 42ºC for 5 min. and 16ºC for 5 min.


  7. End Soak: Incubate at 60°C for 5 minutes, before transformation.

  8. Chill on ice.

  9. Use 2 μl of the reaction to transform 50 μl of competent cells. If reaction will not be used immediately for transformation, store at -20°C.