Agilent Lightning Mutagenesis Primer Design Tool

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 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 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.

Kit Components

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration

Advantages and Features

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

FAQs

Protocols

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

manualE0554

Overview of the Q5^® Site-Directed Mutagenesis Kit
Troubleshooting tips for Q5 ^® Site-Directed Mutagenesis Kit
Quick Tips - How can I improve the efficiency of the DpnI digestion?
Quick Tips - Why do I have so many wildtype colonies?
Quick Tips - Can I make multiple mutations with the Q5 ^® site-directed mutagenesis kit?
Quick Tips - How do I determine the best annealing temperature for my primers?

Quality Control Assays

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.

Certificate of Analysis

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]

E0554S_v2

The following link is to a PDF Safety Data Sheet (SDS) that applies to this product to help you use it safely.

Legal and Disclaimers

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@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^® and Q5^® are registered trademarks of New England Biolabs, Inc.
NEBASECHANGER^™ is a trademark of New England Biolabs, Inc.

Agilent Lightning Mutagenesis Primer Design Tool

Source: https://www.neb.uk.com/product/e0554

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