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  • FAQ: How can I increase transformation efficiency?

    Addition of β-Mercaptoethanol (β-ME) to a final concentration of 24 mM has been shown to increase the transformation efficiency of NEB 5-alpha by 140%. The effect on transformation efficiency may be different when using plasmids other than pUC19. Be sure to use high purity, sterile β-ME at a stock concentration of 1.5 M. Follow the procedure below:
    1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
    2. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
    3. Add 0.8 ìl of 1.5 M β-ME to 50 ìl of cells. Carefully flick the tube 4-5 times to mix cells and β-ME. Do not vortex.
    4. Incubate on ice for 10 minutes.
    5. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
    6. Place the mixture on ice for 30 minutes. Do not mix.
    7. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
    8. Place on ice for 5 minutes. Do not mix.
    9. Pipette 950 µl of room temperature SOC into the mixture.
    10. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
    11. Warm selection plates to 37°C.
    12. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
    13. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours.