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  • Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) Quick Protocol (E0552)

    Primers should be designed with 5´ ends annealing back-to-back. We recommend using the NEB online design software, NEBaseChanger™.

    Protocol

    Step 1: Exponential Amplification
      25 μl RXN FINAL CONC.
    Q5 Hot Start High-Fidelity 2X Master Mix 12.5 μl 1X
    10 μM Forward Primer 1.25 μl 0.5 μM
    10 μM Reverse Primer 1.25 μl 0.5 μM
    Template DNA (1–25 ng/μl) 1 μl 1-25 ng
    Nuclease-free water 9.0 μl  

    Cycling Conditions:
    STEP TEMP TIME
    Initial Denaturation 98°C 30 seconds
    25 Cycles 98°C 10 seconds
    50–72°C* 10–30 seconds
    72°C 20–30 seconds/kb
    Final Extension 72°C 2 minutes
    Hold 4–10°C  
    * For mutagenic primers, please use the Ta provided by the online NEB primer design software, NEBaseChanger™.

    Step 2: KLD Reaction
      VOLUME FINAL CONC.
    PCR Product 1 μl  
    2X KLD Reaction Buffer 5 μl 1X
    10X KLD Enzyme Mix 1 μl 1X
    Nuclease-free Water 3 μl  
    Incubate for 5 minutes at room temperature.

    Step 3: Transformation
    1. Add 5 μl of KLD mix to 50 μl of chemically-competent E. coli cells.
    2. Incubate on ice for 30 minutes.
    3. Heat shock at 42°C for 30 seconds.
    4. Incubate on ice for 5 minutes.
    5. Add 950 μl SOC, gently shake at 37°C for 1 hour.
    6. Spread 40–100 μl onto appropriate selection plate, incubate overnight at 37°C.