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  • Mutagenesis Protocol for Phusion Site-Directed Mutagenesis Kit (F-541)

    Protocol

    1. PCR
      1. Carefully mix and centrifuge all tubes before opening to improve recovery.
      2. Prepare a master mix for the desired number of samples to be mutagenized.
      3. Set up reaction according to Table 1a:
        Table 1a. Pipetting instructions for the mutagenesis reaction (add items in
        this order).
      4. Set up control reaction according to Table 1b: 
        Table 1b. Pipetting instructions for the control reaction (add items in this order).
      5. Phusion Hot Start DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. 25 cycles are recommended for optimal efficiency, and reaction conditions are outlined in Tables 2a and 2b.
        Table 2a. Cycling instructions for the mutagenesis reaction.

        Table 2b. Cycling instructions for the control reaction.
      6. PCR product can be stored at -20°C. Amplification success can be verified by running a 5 µl sample on an agarose gel.
    2. Ligation (5 minute reaction using Quick Ligation™ Kit (NEB# M2200)
      1. Adjust volume of 25ng of PCR product (usually equivalent to 1-5 µl of PCR reaction) to 5 µl with H2O.
      2. Add 5 µl of 2X Quick Ligation Reaction Buffer and mix.
      3. Add 0.5 µl of Quick T4 DNA Ligase and mix thoroughly.
      4. Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.
      5. Chill on ice, then transform or store at -20°C. Do not heat inactivate as this will dramatically reduce transformation efficiency.
    3. Transformation
        Follow standard transformation protocols using an E.coli strain suitable for DNA cloning. Transform 1-10 µl of the reaction mixture per 50-100 µl chemically competent cells. Prior to electroporation, spin column purify the reaction to reduce the PEG concentration.
    4. Analysis
      1. The high efficiency rate of the Phusion Site-Directed Mutagenesis Kit (over 80%) means that mutants can be screened by direct sequencing.
      2. The efficiency of the control reaction can be estimated by the number of blue (mutated) colonies divided by the total number of white (unmutated) colonies when plated on LB-Amp agar plates containing X-Gal and IPTG (over 90%).