Cas9 Nuclease, S. pyogenes

Catalog # Concentration Size
M0386M 20 µM 2500 pmol
M0386S 1000 nM 90 pmol
M0386T 20 µM 500 pmol
Catalog # Concentration Size
M0386M 20 µM 2500 pmol
M0386S 1000 nM 90 pmol
M0386T 20 µM 500 pmol
 
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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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Cas9 Nuclease, S. pyogenes M0386SVIAL -20 1 x 90 pmol 1 µM
  NEBuffer™ r3.1 B6003SVIAL -20 1 x 1.25 ml 10 X
  Cas9 Nuclease, S. pyogenes M0386TVIAL -20 1 x 500 pmol 20 µM
  NEBuffer™ r3.1 B6003SVIAL -20 1 x 1.25 ml 10 X
  Cas9 Nuclease, S. pyogenes M0386MVIAL -20 1 x 2,500 pmol 20 µM
  NEBuffer™ r3.1 B6003SVIAL -20 2 x 1.25 ml 10 X

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

Notes
  • 1,000 nM is equal to 159 ng/μl.
  • 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
  • Jinek M. et al. (2012). Science. 816-821 doi: 10.1126/Science.1225829. Epub 2012 Jun 28.. PubMedID: 22745249
Additional Citations
  • Haussmann IU, Ustaoglu P, Brauer U, Hemani Y, Dix TC, Soller M. (2018) Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4 Nucleic Acids Res Dec 12,PubMedID: 30541104, DOI: 10.1093/nar/gky1254
  • Lee, NC., Larionov, V., Kouprina, N. (2015) Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast Nucleic Acids Res 43(8), e55.PubMedID: 25690893
Publications
  • Pardee P, Green A A, Takahashi M K, Braff D, Lambert G, Lee J W, Ferrante T, Ma D, Donghia N, Fan M, Daringer N M, Bosch I, Dudley D M, O’Connor D H, Gehrke H, Collins J J (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components Cell. DOI: 10.1016/j.cell.2016.04.059
Quality Control Assay
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]
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