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
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. Kit is available with competent cells (NEB #E0554)
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:
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
Storage Note:
The Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) is stable at –20°C for two years. For flexibility, the mutagenesis reagents and control reactions are supplied without competent cells so that any chemically-competent E. coli cells suitable for cloning may be used.
References
Kalnins et al. (1983). The EMBO Journal. 2, 593-597.
Other chemically competent E. coli strains suitable for cloning can be substituted. Results will vary according to the quality and efficiency of the cells.
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 pUC19 DNA (NEB #N3041) (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).
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 E. coli cells have been stored at –80°C.
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.
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 & Change Notifications
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]
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]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
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.
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.
For additional information or to inquire about commercial use, please contact busdev@neb.com.
Trademarks
NEW ENGLAND BIOLABS®, Q5® and SHUFFLE® are registered trademarks of New England Biolabs, Inc. NEBASECHANGER™ is a trademark of New England Biolabs, Inc.
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