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

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 — 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.)

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 — 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

M0386S -20

	Cas9 Nuclease, S. pyogenes	M0386SVIAL	-20	1 x 90 pmol	1 µM
	NEBuffer™ r3.1	B6003SVIAL	-20	1 x 1.25 ml	10 X

M0386T -20

	Cas9 Nuclease, S. pyogenes	M0386TVIAL	-20	1 x 500 pmol	20 µM
	NEBuffer™ r3.1	B6003SVIAL	-20	1 x 1.25 ml	10 X

M0386M -20

	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 Âµg/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

Companion Products

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

Protocols

FAQs

Citations

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

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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

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