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FspEI

Catalog # Concentration Size List Price Quantity Your Price
R0662S 5000 units/ml 200 units
Please Inquire
Catalog # Size List Price Your Price
R0662S 200 units
Please Inquire
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
  • 100% activity in rCutSmart™ Buffer (over 210 enzymes are available in the same buffer) allowing for easier double digests

  • Specificity to epigenetically-relevant DNA modifications (5-mC and 5-hmC)

  • Restriction Enzyme Cut Site: CC(12/16)

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FspEI, an EpiMark® validated product, is a modification-dependent endonuclease which recognizes CmC sites and generates a double-stranded DNA break on the 3´ side of the modified cytosine at N12/N16. Recognized cytosine modifications include C5-methylation (5-mC) and C5-hydroxymethylation (5-hmC) (1). 

This enzyme is provided with an Enzyme Activator Solution which may be used for efficient digestion by FspEI.

The most common epigenetic modifications found in eukaryotic organisms are methylation marks at CpG or CHG sites. A subset of these modified sites are recognized and cleaved by FspEI. 

At fully methylated CpG sites: 
5´. . . C mC  G G . . . 3´
3´. . . G  G mC C . . . 5´

or CHG sites: 
5´. . . C mC H  G G . . . 3´
3´. . . G  G D mC C . . . 5´

H = A or C or T (not G)
D = A or G or T (not C) 

FspEI recognizes each hemi-methylated site individually and cleaves bidirectionally to generate 32 base or 31 base fragments, respectively. These fragments contain the central methylated site and have 4-base 5´ overhangs at each end. FspEI does not cleave unmodified DNA.
Product Source
An E. coli strain that carries the synthetic FspEI gene from Frankia species EAN1pec.

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 (dcm+) DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

Reaction Conditions

1X rCutSmart™ Buffer
Supplement with 1X Enzyme Activator Solution
Incubate at 37°C

1X rCutSmart™ Buffer
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Activity in NEBuffers

NEBufferâ„¢ r1.1: <10%>
NEBufferâ„¢ r2.1: <10%>
NEBufferâ„¢ r3.1: <10%>
rCutSmartâ„¢ Buffer: 100%

Diluent Compatibility

Storage Buffer

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

Heat Inactivation

80°C for 20 minutes

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive



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Notes
  • This enzyme has been used for epigenetic analysis in which target DNA containing CpG sites methylated in both strands is cleaved by FspEI to generate 32 bp fragments containing the methylated CpG sites. The isolated 32 bp fragments can be sequenced to reveal 5-mC modification sites (Cohen-Karni et al., (2011) PNAS 108: 11040-11045).
  • Use of excess enzyme inhibits cleavage. Optimization of the amount of enzyme needed for complete digestion may be required for each substrate DNA.
  • Star activity, including digestion of non-methylated DNA substrates, may result from non-optimal reaction conditions such as extended digestion time, high enzyme or activator concentration or a glycerol concentration of >5%. Optimization of the amount of enzyme needed to achieve efficient specific cuffing, while avoiding star activity, may be required for each substrate DNA.
  • Optimal specificity of cleavage at 5-mC sites can be achieved by keeping the molar ratio of FspEI to 5-mC target sites between 0.1:1 to 1:1 in the presence of 1X Enzyme Activator Solution and digestion for up to 60 minutes. (The molarity of FspEI~0.6 μM).
  • Not sensitive to CpG, dcm, or dam methylation.
References
  • Zheng, Y. et al. (2010). Nucl. Acids Res. doi:10, 1093/nar/gkq327.
  • U.S. Publication No. 2010-0167942 Unpublished observation
  • Cohen-Karni D et al. (2011). The MspJI family of modification dependent restriction endonucleases for epigenetic studies.. Proc. Natl. Acad. Sci. U.S.A.. Jul 5;108(27):, 11040-5.
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]
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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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.

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