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  • Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554)

    Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 

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


    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:
    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
    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 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.