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  • Gibson Assembly® Master Mix – Transformation

    Protocol

    Chemically Competent Cells Transformation Protocol:
    1. Thaw chemically competent cells on ice.
    2. Transfer 50 μl of competent cells to a 1.5 ml microcentrifuge tube (if necessary).
    3. If the chemically competent cells are from New England Biolabs, add 2 μl of assembled product to NEB competent cells and go to step 4 directly. If competent cells are purchased from other manufacture, dilute assembled products 4-fold with H2O prior to transformation. This can be achieved by mixing 5 μl of assembled products with 15 μl of H2O. Add 2 μl of the diluted assembled product to competent cells.
    4. Mix gently by pipetting up and down or flicking the tube 4–5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.
    5. Heat shock at 42°C for 30 seconds.* Do not mix.
    6. Transfer tubes on ice for 2 minutes.
    7. Add 950 μl of room temperature SOC media* to tubes.
    8. Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
    9. Warm selection plates to 37°C.
    10. Spread 100 μl of the cells onto the plates with appropriate antibiotics. Use Amp plates for positive control sample.
    11. Incubate plates overnight at 37°C.

      * Please note: Follow the manufacturer's protocols for the duration and temperature of the heat shock step, as well as the optimal medium for recovery. Typically, transformation of our positive control assembly product will yield more than 100 colonies on an Amp plate with greater than 80% colonies containing inserts.
    Electrocompetent Cells Transformation Protocol:
    1. Thaw electrocompetent cells on ice.
    2. Transfer 50 μl of electrocompetent cells to a pre-chilled electroporation cuvette with 1 mM gap.
    3. Dilute assembled products 3-fold with H2O prior to electroporation. This can be achieved by mixing 5 μl of assembled products with 10 μl of H2O. Add 1 μl of the diluted assembly product to electrocompetent cells. Mix gently by pipetting up and down or flicking the tube 4–5 times.
    4. Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells.Add 950 μl of room temperature SOC media* to tubes.
    5. Add 950 μl of room temperature SOC media to the cuvette immediately after electroporation.
    6. Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
    7. Warm selection plates to 37°C.
    8. Spread 100 μl of the cells onto the plates.
    9. Incubate overnight at 37°C.