EnGen® sgRNA Synthesis Kit, S. pyogenes
Product informationCode | Name | Size | Quantity | Price | |
---|---|---|---|---|---|
E3322V |
EnGen sgRNA Synthesis Kit, S. pyogenes |
10 rxns | - | Unavailable in your region | |
E3322S |
EnGen sgRNA Synthesis Kit, S. pyogenes |
20 rxns | - | Unavailable in your region |
EnGen® sgRNA Synthesis Kit, S. pyogenes
Note: Kit and protocol have been updated. Please visit Protocols & Manuals tab for details.
Product Introduction
The EnGen sgRNA Synthesis Kit, S. pyogenes provides a simple and quick method for transcribing high yields of sgRNA in a single 30-minute reaction, using the supplied reagents and target-specific DNA oligos designed by the user.
- Rapid generation of microgram quantities of sgRNA
- Complete workflow of less than one hour
- Simple protocol requires only a user-supplied ~55 nucleotide ssDNA target-specific oligonucleotide
- For help with oligo design, try the EnGen sgRNA Template Oligo Designer
- Compatible with EnGen Spy Cas9 NLS, EnGen Spy Cas9 Nickase, EnGen Spy dCas9 (SNAP-tag®)
Catalog # | Size | Concentration |
---|---|---|
E3322V | 10.0 reactions | |
E3322S | 20.0 reactions |
- Product Information
- Protocols, Manuals & Usage
- Tools & Resources
- FAQs & Troubleshooting
- Citations & Technical Literature
- Quality, Safety & Legal
- Other Products You May Be Interested In
Product Information
Description
The EnGen sgRNA Synthesis Kit, S. pyogenes provides a simple and quick method for transcribing high yields of sgRNA in a single 30 minute reaction, using the supplied reagents and target-specific DNA oligos designed by the user.
In nature, S. pyogenes Cas9 is programmed with two separate RNAs, the crRNA and tracrRNA. The crRNA, or CRISPR RNA sequence contains approximately 20 nucleotides of homology complementary to the strand of DNA opposite and upstream of a PAM (Protospacer Adjacent Motif) (NGG) sequence. The tracrRNA, or transactivating crRNA, contains partial complementary sequence to the crRNA as well as the sequence and secondary structure that is recognized by Cas9. These sequences have been adapted for use in the lab by combining the tracrRNA and crRNA into one long single guide RNA (sgRNA) (1) species capable of complexing with Cas9 to recognize and cleave the target DNA.
The EnGen sgRNA Synthesis Kit, S. pyogenes combines an S. pyogenes Cas9-specific Scaffold Oligo (included in the EnGen 2X sgRNA Reaction Mix) that is partially complementary to the target-specific oligos designed by the user. The two oligos anneal at the overlapping region and are filled in by the DNA polymerase, creating a double-stranded DNA (dsDNA) template for transcription. Synthesis of the dsDNA template and transcription of RNA occur in a single reaction, resulting in the generation of a functional sgRNA.
1. Jinek, M. et al. (2012) Science 816–821. PubMed ID: 22745249.
- This product is related to the following categories:
- sgRNA Synthesis Reagents and Kits Products,
- Genome Editing 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:
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Properties & Usage
Materials Required but not Supplied
Target-specific DNA oligo(s)Thermocycler/37°C heat block/incubator
Nuclease-free water
Equipment and reagents for RNA quantitation
Spin columns for RNA cleanup (e.g. NEB #T2040)
RNase-free tubes, aerosol tips
Microcentrifuge
Optional Materials:
Alkaline Phosphatase, Calf Instestinal (CIP)
Cas9 Nuclease, S. pyogenes
EnGen Spy Cas9, NLS
Gels, running buffer, gel loading dye, RNA and DNA ladders, gel box
Related Products
Companion Products
Protocols, Manuals & Usage
Protocols
Manuals
The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.
Tools & Resources
Web Tools
FAQs & Troubleshooting
FAQs
- The sgRNA is not running as a single band on the gel – why is this happening?
- Is it necessary to add a “G” into the oligo following the T7 promoter sequence?
- Is it necessary to DNase treat my sgRNA synthesis reaction?
- Why is my sgRNA not exhibiting 100% in vitro Cas9 cleavage activity?
- Is it necessary to phosphatase treat my sgRNAs?
- The sgRNA is not running as a single band on the gel – why is this happening?
- Will the sgRNAs synthesized from this kit (NEB #E3322S) be recognized by homologs of Cas9 from different organisms?
- Do I include the PAM(NGG) in my sgRNA sequence?
- What is the sequence of the control oligo provided with the EnGen sgRNA Synthesis Kit, S. pyogenes?
- Is the Monarch Spin RNA Cleanup Kit (NEB #T2040) compatible with the EnGen sgRNA Synthesis Kit, S. pyogenes?
- Is it necessary to add the provided DTT to the sgRNA synthesis reaction?
Citations & Technical Literature
Citations
Additional Citations
- Evan A. Boyle, Johan O.L. Andreasson, Lauren M. Chircus, Samuel H. Sternberg, Michelle J. Wu, Chantal K. Guegler, Jennifer A. Doudna, and William J. Greenleaf (2017) High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding Proc Natl Acad Sci U S A; 114, 5461-5466.
Publications
- Gertsenstein, M., & Nutter, L. M. J. (2018) Engineering point mutant and epitope-tagged alleles in mice using Cas9 RNA-guided nuclease Curr Protoc Mouse Biol;
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]- E3322S_v1_10012893
- E3322S_v1_10013330
- E3322S_v1_10022304
- E3322S_v1_10034126
- E3322S_v2_10034536
- E3322S_v2_10044273
- E3322S_v2_10041543
- E3322S_v2_10050946
- E3322S_v2_10065248
- E3322V_v2_10068949
- E3322S_v2_10078948
- E3322V_v2_10083641
- E3322S_v2_10099777
- E3322V_v2_10100206
- E3322S_v2_10120188
- E3322S_v2_10125379
- E3322S_v2_10130633
- E3322V_v2_10132945
- E3322S_v2_10154218
- E3322V_v2_10161539
- E3322S_v2_10175382
- E3322V_v2_10185455
- E3322S_v2_10209939
- E3322V_v2_10211731
- E3322S_v1_0051709
- E3322V_v2_10220481
- E3322S_v2_10222371
- E3322V_v2_10231015
- E3322V_v2_10236650
- E3322V_v2_10237140
- E3322S_v2_10239220
- E3322S_v2_10242329
- E3322S_v2_10254262
- E3322V_v2_10254015
- E3322S_v2_10260307
Safety DataSheets
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.EnGen® sgRNA Enzyme Mix
EnGen® 2X sgRNA Reaction Mix, S. pyogenes
DNase I (RNase-free)
EnGen® sgRNA Control Oligo, S. pyogenes
Dithiothreitol (DTT)
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.
Other Products You May Be Interested In
The supporting documents available for this product can be downloaded below.