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EnGen® Mutation Detection Kit

Catalog # Concentration Size List Price Quantity Your Price
E3321S 25 reactions $342.00
$307.80
Catalog # Size List Price Your Price
E3321S 25 reactions $342.00
$307.80
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
  • Simple protocol for detecting targeting efficiency in genome editing experiments
  • Optimized reagents for performing robust T7 Endonuclease-based detection of genome editing events
  • Q5® Hot Start High Fidelity 2X Master Mix included for robust amplification, high fidelity and convenience
  • Rapid protocol requires no cleanup between PCR and digestion






The EnGen Mutation Detection Kit provides reagents for detection of on-target genome editing events. In the first step, targeted regions from cells whose genomes were targeted (i.e. CRISPR/Cas9, TALENs, Zinc-finger Nucleases) are amplified using Q5 Hot Start High-Fidelity 2X Master Mix. Upon denaturation and re-annealing, heteroduplexes are formed when mutations from insertions and deletions (indels) are present in the amplicon pool. In the second step, annealed PCR products are digested with EnGen T7 Endonuclease I, a structure-specific enzyme that will recognize mismatches larger than 1 base. Both strands of the DNA are cut when a mismatch is present, which results in the formation of smaller fragments. Analysis of the resulting fragments provides an estimate of the efficiency of the genome editing experiments.

The EnGen Mutation Detection Kit includes a Control Template and Primer Mix that can be used as a control for the PCR reaction and T7 Endonuclease I digestion. The Control Template and Primer Mix provided contains two plasmids and primers that when amplified, denatured and re-annealed will form heteroduplexes that contain a 10-base insertion. This structure is a substrate for T7 Endonuclease I. The digestion of the 600 bp heteroduplex containing amplicon yields products of 200 bp and 400 bp. 600 bp parental homoduplexes are uncleaved, and are easily distinguished from cleaved heteroduplexes when separated and visualized by agarose gel electrophoresis or fragment analysis instrument.

The protocol has been optimized so that PCR products generated by the Q5 Hot Start High-Fidelity 2X Master Mix can be introduced directly into the T7 Endonuclease I digestion without the need for purification. Digestion of the heteroduplex is complete in only 15 minutes, and Proteinase K is included to stop the reaction efficiently. Additional Q5 Hot Start High-Fidelity 2X Master Mix is also included to allow for optimization of target site amplification before digestion.

Figure 1: Workflow for Mutation Detection Kit


Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding three classes of possible structures. Duplexes containing a mismatch greater than one base are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to estimate targeting efficiency.
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 M0494AAVIAL -20 1 x 0.4 ml 2 X
  NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
  Control Template and Primer Mix S0534AVIAL -20 1 x 0.025 ml 10 X
  Proteinase K, Molecular Biology Grade P8107AVIAL -20 1 x 0.12 ml 800 units/ml
  Quick-Load® Purple 1 kb Plus DNA Ladder N0550AVIAL 4 1 x 0.2 ml 100 µg/ml
  Gel Loading Dye, Purple (6X), no SDS B7025SVIAL 25 1 x 1 ml 6 X
  EnGen® T7 Endonuclease I M0450AVIAL -20 1 x 0.025 ml

Properties & Usage

Materials Required but not Supplied

Oligodeoxyribonucleotide primers for PCR
PCR reaction tubes or strips
Nuclease-free water
Thermocycler
Aerosol tips for PCR

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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

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.

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.

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