Product Class: Other

Cas9 Nuclease, S. pyogenes
icon_cloned icon_recomb icon_neb3_1 icon_incTemp37
Item# Description List Price Web Price Qty
M0386M Cas9 Nuclease, S. pyogenes - 2,000 pmols (concentration 20 uM) $767.00
$690.30
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M0386S Cas9 Nuclease, S. pyogenes - 70 pmols $71.00
$63.90
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M0386T Cas9 Nuclease, S. pyogenes - 400 pmols (concentration 20 uM) $192.00
$172.80
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
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EnGen™ Spy Cas9 NLS NEB #M0646 is also available now. 

    Product Introduction

     

    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

    An E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    NEBuffer™ 3.1 -20 10X
    Product Categories:
    Genome Editing Products,
    Nucleases Products
    Applications:
    Genome Editing Applications

    Properties & Usage

      Reaction Conditions

      1X NEBuffer™ 3.1
      Incubate at 37°C

      1X NEBuffer™ 3.1
      100 mM NaCl
      50 mM Tris-HCl
      10 mM MgCl2
      100 µg/ml BSA
      (pH 7.9 @ 25°C)

      Storage Temperature

      -20°C

      Storage Conditions

      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 min

      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

      Datacards

      The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)

      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?

      Troubleshooting

      Quality & Safety

      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

      This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

      While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

      For more information about commercial rights, please contact NEB's Global Business Development team at [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.