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  • Transformation Protocol (M0369)

    Electrocompetent strains of E. coli (commercially available or prepared by user) can be transformed by ligation products prepared with ElectroLigase. Chemically competent cells are also compatible, but for maximum performance with chemically competent cells, please consider using the Blunt/TA Ligase Master Mix (NEB #M0367). The following protocol is recommended by NEB. Other protocols can be used but the volume of ligation reaction used should not exceed 5 μl reaction per 50 μl cells.

    Protocol

    1. Thaw competent cells on ice.

    2. Aliquot 40 μl of cells into a 1.5 ml microcentrifuge tube on ice.

    3. Add 2 μl of the ligation reaction to the cells and mix by finger-flicking. Do not vortex the tube.

    4. Transfer DNA/competent cell mixture to a prechilled electroporation cuvette and follow the manufacturers recommendations for electroporation (e.g. 2500 V, 200 Ω, 25 μF, 2 mm gap cuvette).

    5. Add 760 μl recovery media (e.g. SOC) to the cuvette, mix, transfer the transformed cells to a culture tube and incubate for one hour at 37°C with shaking (200–250 rpm).

    6. Spread 50 μl of the outgrowth (undiluted or diluted 1:5 with recovery media) onto appropriate antibiotic selection plates and incubate overnight at 37°C.

    7. Typical Results:
      Transformation efficiencies around 3 x 106 cfu/μg are typically achieved for recombinant blunt-end vectors (vector + insert), using cells with a 5 x 109 calculated efficiency with uncut DNA. Results for TA cloning and standard cohesive end (4 bp overhang) cloning produce even higher numbers, often over 107 cfu/μg. This corresponds to several hundred colonies on a plate when 50 μl of the outgrowth is plated at a 1:5 dilution. As with all ligation and transformation protocols, many factors affect the calculated transformation efficiency, including purity and integrity of DNA ends, competence of the cells being transformed, media choices, incubation temperatures and times and biological effects (intact ORF in high-copy vector, toxic genes, etc.).