Product Class: Other

Cas9 Nuclease, S. pyogenes
neb31 cloned at NEB recombinant 37 65 Heat

Now available: EnGen Spy Cas9 HF1 NEB #M0667 for reduction of off-target cleavage

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.

Product Introduction

 
Catalog # Size Concentration
M0386S 90.0 pmol 1000 nM
M0386T 500.0 pmol 20 µM
M0386M 2500.0 pmol 20 µM

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Product Information

Description








    
Cas9 Nuclease, S. pyogenes, is an RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA. The location of the break is within the target sequence 3 bases from the NGG PAM (Protospacer Adjacent Motif) (1). The PAM sequence, NGG, must follow the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence.




Cas9 in vivo: Bacterial Adaptive Immunity Cas9 in vivo: Bacterial Adaptive Immunity

CRISPR loci in the bacterial genome are transcribed and processed into crRNA. Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)


Cas9 nuclease, S. pyogenes, complexed with an sgRNA Schematic representation of Cas9 Nuclease, S. pyogenes sequence recognition and DNA cleavage
Cleavage occurs three nucleotides upstream of the PAM sequence (shown in red). sgRNAs are complimentary to the strand of DNA opposite of the PAM.


In vitro DNA cleavage using Cas9 Nuclease, S. pyogenes In Vitro DNA cleavage using Cas9 Nuclease, S. pyogenes

Reactions were set up according to recommended conditions, and cleavage products were resolved on a 1% agarose TBE gel. Input DNA is PvuII-linearized pBR322 plasmid DNA.

Product Source

A His-tagged engineered variant of E. coli that carries the cloned Cas9 gene from Streptococcus pyogenes.
This product is related to the following categories:
Genome Editing Products,
Programmable Nucleases Products,
This product can be used in the following applications:
Genome Editing Applications

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

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

Properties & Usage

Reaction Conditions

1X NEBuffer™ r3.1
Incubate at 37°C

1X NEBuffer™ r3.1
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Storage Buffer

300 mM NaCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 5 minutes

Product Notes

  1. 1,000 nM is equal to 159 ng/μl.
  2. If planning to use higher concentration Cas9 Nuclease, S. pyogenes (M0386T and M0386M) for in vitro digestion of DNA, the enzyme should be diluted to 1 µM using Diluent B (NEB #B8002S) prior to the reaction assembly.

References

  1. Jinek M. et al. (2012). Science. 816-821 doi: 10.1126/Science.1225829. Epub 2012 Jun 28.. PubMedID: 22745249

Protocols, Manuals & Usage

Protocols

  1. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
  2. Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)

Application Notes

FAQs & Troubleshooting

FAQs

  1. Why do I observe incomplete digestion?
  2. Why does digestion efficiency differ between two SgRNAs?
  3. Does NEB provide plasmids for gRNA cloning?
  4. Does Cas9 Nuclease contain a nuclear localization signal (NLS)? 
  5. Is Cas9 Nuclease fused with a N-terminal 6XHis tag?
  6. How do I dilute the enzyme to 1 μM for in vitro reactions?
  7. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

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 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.

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