Product Class: Kit

EnGen® Mutation Detection Kit

Product Introduction

  • 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
Catalog # Size Concentration
E3321S 25.0 reactions

Product Information

Description







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.
This product is related to the following categories:
Mutation Detection Reagents and Kits Products,
Genome Editing Products,
DNA Repair Enzymes and Structure-specific Endonucleases Products,
This product can be used in the following applications:
Genome Editing Applications

Kit Components

Kit Components

The following reagents are supplied with this product:

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

Properties & Usage

Materials Required but not Supplied

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

Protocols, Manuals & Usage

Protocols

  1. T7 Endonuclease I-based Mutation Detection with the EnGen® Mutation Detection Kit (NEB #E3321)
  2. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX

Manuals

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

Application Notes

FAQs & Troubleshooting

FAQs

  1. Why do I see an extra band when I run the undigested heteroduplex on an agarose gel?
  2. Why do I see very little DNA on my gel or low signal on the fragment analyzer?
  3. Will EnGen® T7 Endonuclease I recognize single base mismatches and single base insertions or deletions (indels)?  
  4. Can cell lysates be used in the PCR reaction?
  5. What are the expected results using the included control?
  6. My PCR reaction yield is low, can I add more than 5 µl of the PCR reaction to the digestion reaction?

Troubleshooting

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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 [email protected].

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 [email protected].

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