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Q5® Site-Directed Mutagenesis Kit

Catalog # Concentration Size List Price Quantity Your Price
E0554S 10 reactions $325.00
$292.50
Catalog # Size List Price Your Price
E0554S 10 reactions $325.00
$292.50
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

The Q5® Site-Directed Mutagenesis Kit 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 set up
  • 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
  • Supplied with competent cells 
  • Use NEBaseChanger™ tool to generate primer sequences and an annealing temperature
Featured Videos
View Video Library
  • Monarch_MiniprepProtocol_VideoThumb
    NEBaseChanger® Primer Design Tool Tutorial
  • Monarch_MiniprepProtocol_VideoThumb
    Quick Tips - How can I improve the efficiency of the DpnI digestion?
  • Monarch_MiniprepProtocol_VideoThumb
    Overview of the Q5® Site-Directed Mutagenesis Kit
  • Monarch_MiniprepProtocol_VideoThumb
    Quick Tips - Why do I have so many wildtype colonies?
  • Monarch_MiniprepProtocol_VideoThumb
    Quick Tips - Can I make multiple mutations with the Q5® site-directed mutagenesis kit?
  • Monarch_MiniprepProtocol_VideoThumb
    Troubleshooting tips for Q5® Site-Directed Mutagenesis Kit
The Q5 Site-Directed Mutagenesis Kit 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 NEB 5-alpha Competent E. coli, provided with the kit, 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 Kit Overview. Figure 2: Q5 Site-Directed Mutagenesis Kit 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 (provided).
Figure 3: Primer Design for the Q5 Site-Directed Mutagenesis KitFigure 3: Primer Design for the Q5 Site-Directed Mutagenesis Kit

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 back-to- 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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Q5® Hot Start High-Fidelity 2X Master Mix M0494AVIAL -20 1 x 0.125 ml 2 X
  KLD Reaction Buffer B0554AVIAL -20 1 x 0.15 ml 2 X
  KLD Enzyme Mix M0554AVIAL -20 1 x 0.01 ml 10 X
  Control SDM Primer Mix S0554AVIAL -20 1 x 0.01 ml 10 µM
  Control SDM Plasmid N0554AVIAL -20 1 x 0.01 ml 5 µg/ml
  pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl
  SOC Outgrowth Medium B9020SVIAL 4 1 x 25 ml
  NEB® 5-alpha Competent E. coli (High Efficiency) C2987HVIAL -80 10 x 0.05 ml
Features
  • Generation of mutations, insertions or deletions in plasmid DNA
  • 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 set-up
  • 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

Properties & Usage



Companion Products
Notes
  • Storage Note:
    The Q5 Site-Directed Mutagenesis Kit is stable at –80°C for one year. For convenience, the Q5 Hot Start High-Fidelity 2X Master Mix, KLD Enzyme Mix, KLD Reaction Buffer, Control Primers and Template DNA are packaged together in a separate box that can be removed and stored at –20°C for two years with no loss of activity. The SOC can be removed and stored at room temperature. It is important to store the NEB 5-alpha Competent E. coli at –80°C, and avoid repeated freeze-thaw cycles.
References
  • Kalnins et al., (1983). The EMBO Journal. 2, 593-597.
  • Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. (2013). Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.. Nat Methods. Sep 1, PubMedID: 23995389
Web Tools
FAQs
Tech Tips
  • 1. If resulting plasmids do not contain the desired mutation (wild-type sequence), we recommend using ≤ 10 ng of template in the PCR step. Alternatively, the background wild-type plasmids can be reduced by increasing the KLD incubation time to 30-60 minutes.

    2. If there are no or low colonies, ensure that your primers are designed properly. To take advantage of the exponential nature of the amplification reaction, the 5´ ends of the two primers should align back-to-back unless deletions are being made. For best results, primers should be designed and annealing temperatures calculated using NEBaseChanger™, the NEB online primer design software.

    3. If there is no or low PCR product, ensure that the optimal annealing temperature (Ta) is used. High-Fidelity polymerases benefit from a Tm+3 annealing temp. Use NEBaseChanger™, the NEB online primer design software, to calculate Ta. Alternatively, the optimal annealing temperature could be determined using a gradient PCR followed by agarose gel analysis.
Citations
Additional Citations
  • Yafeng Li, Delu Song, Ying Song, Liangliang Zhao, Natalie Wolkow, John W Tobias, Wenchao Song, Joshua L Dunaief (2015) Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium. J Biol Chem 290, 11918-34.PubMedID: 25802332, DOI: 10.1074/jbc.M115.645903
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
Legal And Disclaimer

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

This product is covered by one or more patents.

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

For additional information or to inquire about commercial use, please contact busdev@neb.com.

NEW ENGLAND BIOLABS® and Q5® are registered trademarks of New England Biolabs, Inc.
NEBASECHANGER™ is a trademark of New England Biolabs, Inc.
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