Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)
Product informationCode | Name | Size | Quantity | Price | |
---|---|---|---|---|---|
E0552S |
Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) |
10 rxns | - | Unavailable in your region |
Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)
Available with competent cells (NEB #E0554)
Product Introduction
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
Catalog # | Size | Concentration |
---|---|---|
E0552S | 10.0 reactions |
Featured Videos
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NEBaseChanger® Primer Design Tool Tutorial
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Quick Tips - How can I improve the efficiency of the DpnI digestion?
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Overview of the Q5® Site-Directed Mutagenesis Kit
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Quick Tips - Why do I have so many wildtype colonies?
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Quick Tips - Can I make multiple mutations with the Q5® site-directed mutagenesis kit?
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Troubleshooting tips for Q5® Site-Directed Mutagenesis Kit
- Product Information
- Protocols, Manuals & Usage
- Tools & Resources
- FAQs & Troubleshooting
- Citations & Technical Literature
- Quality, Safety & Legal
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Product Information
Description
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)- This product is related to the following categories:
- DNA Assembly, Cloning and Mutagenesis Kits Products
- This product can be used in the following applications:
- Site Directed Mutagenesis,
- Site Directed Mutagenesis
Kit Components
Kit Components
The following reagents are supplied with this product:
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Properties & Usage
Materials Required but not Supplied
- Chemically-competent E. coli cells
- SOC Outgrowth Media
- Selection Plates
Features
- 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
Related Products
Companion Products
- Q5® Site-Directed Mutagenesis Kit (Without Competent Cells)
- Q5® Site-Directed Mutagenesis Kit
- Q5® Hot Start High-Fidelity 2X Master Mix
- NEB® 5-alpha Competent E. coli (High Efficiency)
- SOC Outgrowth Medium
- Monarch® Plasmid Miniprep Kit
- Monarch® DNA Gel Extraction Kit
- Monarch® PCR & DNA Cleanup Kit (5 μg)
Product Notes
- 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.
Protocols, Manuals & Usage
Protocols
Manuals
Tools & Resources
Web Tools
FAQs & Troubleshooting
FAQs
- What is the maximum number of nucleotides that can be inserted with this kit?
- What is the maximum distance that can be tolerated between substitutions?
- Typically, what percentage of transformants will have the desired mutation incorporated?
- What is the KLD Mix?
- What types of competent cells are compatible with this kit?
- If I double my PCR size, should I add more PCR mix to the KLD reaction?
- Why is the desired mutation missing from the transformants that I screened?
- Why do I not see my PCR product after using the Q5® Site-Directed Mutagenesis Kit?
- What plasmid sizes can be amplified using the Q5® Site-Directed Mutagenesis Kit?
- Do I need to purify my plasmid before or after the KLD reaction when using the Q5® Site-Directed Mutagenesis Kit?
- How do I design primers to use with the Q5® Site-Directed Mutagenesis Kit?
- What should I use for an annealing temperature with the Q5® Site-Directed Mutagenesis Kit?
- I use the Q5 Site-Directed Mutagenesis Kit to introduce single mutations. How can I introduce multiple mutations?
Troubleshooting
No/Low Colonies
- Be sure to use high-efficiency chemically-competent E. coli cells. The following competent E. coli cells have been shown to work with this kit:
NEB #C2987, NEB 5-alpha (High Efficiency) (standard recommendation)
NEB #C2992, NEB 5-alpha F´Iq (High Efficiency)
NEB #C3019, NEB 10-beta (High Efficiency)
NEB #C2984, NEB Turbo
NEB #C2566, T7 Express
NEB #C3029, Shuffle® T7
For convenience, we offer a version of the Q5 Site Directed Mutagenesis Kit prepackaged with
NEB #C2987 NEB 5-alpha Competent E. coli (High Efficiency) cells in product number NEB #E0554.
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.
- 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
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
Additional Citations
Quality, Safety & Legal
Quality Assurance Statement
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]- E0552S_v1_10008841
- E0552S_v1_10016543
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Safety DataSheets
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.KLD Reaction Buffer
Q5® Hot Start High-Fidelity 2X Master Mix
Control SDM Plasmid
Control SDM Primer Mix
KLD Enzyme Mix
Legal and Disclaimers
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.The supporting documents available for this product can be downloaded below.