Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)

The Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours.

  • Non-overlapping primer design ensures robust, exponential amplification, generating a high percentage of desired mutations from a wide range of templates
  • Intramolecular ligation and transformation into NEB high-efficiency competent cells results in high colony yield
  • Extremely low error rate of Q5 Hot Start High-Fidelity DNA Polymerase reduces screening time
  • Hot start polymerase enables room temperature reaction setup
  • DpnI background reduction permits a wide range of starting template concentrations
  • Use of standard primers eliminates additional expenses from phosphorylated or purified oligos
  • Easy-to-use PCR master mix and unique multi-enzyme KLD mix offer convenience and quality
  • Rapid and direct treatment step proceeds at room temperature in 5 minutes
  • Allows the use of any chemically-competent E. coli cells suitable for cloning
  • Use NEBaseChanger to generate primer sequences and an annealing temperature

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  • Product Information
    The Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency chemically-competent E. coli, not supplied, ensures robust results with plasmids up to at least 20 kb in length.

    Figure 1: Site-specific mutagenesis proceeds in less than 2 hours.Figure 1: Site-specific mutagenesis proceeds in less than 2 hours.

    The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most plasmids, the mutagenesis reaction is complete in less than two hours.
       
    Figure 2: Q5 Site-Directed Mutagenesis Overview.Figure 2: Q5 Site-Directed Mutagenesis Overview.

    This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into doublestranded plasmid DNA. The first step is an exponential amplification using standard primers and a master mix fomulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a unique enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemicallycompetent cells (not provided).
    Figure 3: Primer Design for Q5 Site-Directed MutagenesisFigure 3: Primer Design for Q5 Site-Directed Mutagenesis

    Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed forward (black) and reverse (red) primers. Unlike kits that rely on linear amplification, primers designed for the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits of exponential amplification are realized. A) Substitutions are created by incorporating the desired nucleotide change(s) (denoted by *) in the center of the forward primer, including at least 10 complementary nucleotides on the 3´side of the mutation(s). The reverse primer is designed so that the 5´ends of the two primers anneal backto-back. B) Deletions are engineered by designing standard, non-mutagenic forward and reverse primers that flank the region to be deleted. C) Insertions less than or equal to 6 nucleotides are incorporated into the 5´ end of the forward primer while the reverse primer anneals back-to-back with the 5´ end of the complementary region of the forward primer. D) Larger insertions can be created by incorporating half of the desired insertion into the 5´ ends of both primers. The maximum size of the insertion is largely dictated by oligonucleotide synthesis limitations.
    Figure 4: NEB’s Q5 SDM Kit delivers higher transformation efficiency than Agilent’s QuikChange® SDM Kit
    Q5 Graph
    Results from a substitution reaction (4 nt) using the back-to-back Control SDM Primer Mix and Control SDM Plasmid (6.7 kb) are shown, along with results from a 12 nt deletion experiment (5.8 kb plasmid) and an 18 nt insertion experiment (7.0 kb plasmid). In all three cases, over 90% of the resultant colonies had incorporated the desired mutation(s). Results are normalized to total transformants if cells were not diluted prior to plating. For comparison, the same substitution reaction (4 nt) was performed with the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) following Agilent’s protocol and using Agilent’s primer design tool to design overlapping primers.

    *Note that the QuikChange kit does not accommodate deletions and insertions of this size, so no comparison could be made for these experiments.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    KLD Reaction Buffer -20 2 X
    Q5® Hot Start High-Fidelity 2X Master Mix -20 2 X
    KLD Enzyme Mix -20 10 X
    Control SDM Plasmid -20 5 μg/ml
    Control SDM Primer Mix -80 10 μM
    Product Categories:
    DNA Assembly Cloning and Mutagenesis Kits
    Applications:
    Site Directed Mutagenesis,
    Site Directed Mutagenesis
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