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
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
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
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 chemically competent cells (not provided). Figure 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 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.
This product is related to the following categories:
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
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,
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 (see Figure 3). For best results, primers should be designed and annealing temperatures calculated using NEBaseChanger™, the NEB online primer design software.
Ensure there is a clean PCR product by visualizing 2–5 μl of the reaction on an agarose gel. Follow the suggestions below for low or impure PCR products.
Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. If the PCR yield is low, more product can be added to the KLD reaction, however a buffer exchange step, such as PCR purification, must be included prior to transformation.
Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.
Ensure that the selectable marker in the plasmid matches the selection agent used in the plates
Ensure the NEB 5-alpha Competent E. coli cells have been stored at -80° C.
Check that the transformation efficiency of the competent cells is ~1 x 109 colony forming units (cfu) per μg. To calculate transformation efficiency, transform 2 μl of the provided control pUC19 DNA (100 pg) into 50 μl of cells. Follow the transformation protocol on page 8. Prior to plating, dilute 10 μl of cells up to 1 ml in SOC. Plate 100 μl of this dilution. In this case, 150 colonies will yield a transformation efficiency of 1.5 x 109 cfu/μg
(μg DNA=0.0001, dilution=10/1000 x 100/1000).
No/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.
Ensure that the elongation time is adequate for the plasmid length. We recommend 20–30 seconds per kb of plasmid.
Ensure that the final concentration of each primer is 0.5 μm.
Purify the primers with polyacrylamide gel electrophoresis (PAGE).
Resulting Plasmids Do Not Contain the Desired Mutation
Ensure proper design of the mutagenic primers.
Optimize the PCR conditions (see above).
Use 1–25 ng of template in the PCR step. A small increase in the number of clones with no/incorrect mutation incorporated can occur if less than 1 ng or more than 25 ng of template is used.
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 & Technical Literature
Citations
Product Citation Tool
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
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Specifications & Change Notifications
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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.
Licenses
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.
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Trademarks
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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