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Cas9 Nuclease NLS, S. pyogenes
This product has been discontinued on 10/1/16, and replaced by EnGen™ Cas9 NLS, S. pyogenes (NEB #M0646).
This product was discontinued on 10/01/2016
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Description
Related Products
References
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Information
Properties and Usage
Notes
Description
Cas9 Nuclease NLS, 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
Nuclease NLS, S. pyogenes contains a single Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the C terminus of
the protein.
Cas9 nuclease, S. pyogenes, complexed with an sgRNA
Cleavage occurs three nucleotides upstream of the PAM sequence (shown in red). sgRNAs are complimentary to the strand of DNA opposite of
the PAM.
Product Source
An E. coli strain that carries the cloned Cas9 gene from Streptococcus pyogenes with a C-terminal Simian virus 40 (SV40) nuclear
localization signal (NLS) and a N-terminal 6XHis tag.
Reagents Supplied
The following reagents are supplied with this product:
Cas9 Nuclease Reaction Buffer
Store at (°C)
Concentration
-20
10X
Properties and Usage
Reaction Conditions
1X Cas9 Nuclease Reaction Buffer
Incubate at 37°C
1X Cas9 Nuclease Reaction Buffer:
20 mM HEPES
100 mM NaCl
5 mM MgCl2
0.1 mM EDTA
pH 6.5 @ 25°C
Storage Conditions
300 mM NaCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C
Related Products
Companion Products
EnGen® sgRNA Synthesis Kit, S. pyogenes
EnGen® Mutation Detection Kit
Notes
1. 1,000 nM is equal to 160 ng/μl.
References
1. Jinek M. et al. (2012). Science. 816-821. DOI: 10.1126/Science.1225829. Epub 2012 Jun 28, PubMedID: 22745249
FAQs
FAQs
1.
2.
3.
4.
5.
Where is the nuclear localization signal on Cas9 NLS S. pyogenes located?
Which nuclear localization signal is fused to Cas9?
Why do I observe incomplete digestion?
Why does digestion efficiency differ between two SgRNAs?
Does NEB provide plasmids for gRNA cloning?
Datacards
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.
M0641Datasheet-Lot0071507
M0641Datasheet-Lot0071601
Quality Control
Datacards
Safety Data Sheet
Quality Control
Quality Control Assays
The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product.
Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting
Documents section of this page. Further information regarding NEB product quality can be found here.
Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to
the nicked form is determined by agarose gel electrophoresis.
Exonuclease Activity (Radioactivity Release):
The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4
hours the exonuclease activity is determined by the % release of radioactive nucleotides.
Functional Test (Cas9 Nuclease, Targeted Digestion):
Cas9 Nuclease is tested in a reaction containing target DNA and guide RNA. The percent digested target DNA is determined by
agarose gel electrophoresis.
Non-Specific DNase Activity (16 hour):
The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16
hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
Protein Purity (SDS-PAGE):
The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest
band in a sample of known dilution. The purity is determined by SDS-PAGE.
RNase Assay (Extended Digestion):
The product is tested in a reaction containing a fluorescent RNA substrate. After incubation >90% of the substrate RNA remains
intact as determined by gel electrophoresis.
Safety Data Sheet
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
Cas9 Nuclease NLS, S. pyogenes
Cas9 Nuclease Reaction Buffer
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.
M0641Datasheet-Lot0071507
M0641Datasheet-Lot0071601
Legal and Disclaimers
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.