Product Class: Restriction Endonuclease


cloned at neb recombinant icon_CutSmart incubation temp heat inactivation
Catalog #SizeConcentration
R0662S200 units5,000 units/ml
R0662L1,000 units5,000 units/ml


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.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart® Buffer-2010X
Enzyme Activator Solution30X

Properties and 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 CutSmart® Buffer
Supplement with 1X Enzyme Activator Solution
Incubate at 37°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 10%
NEBuffer 2.1: 10%
NEBuffer 3.1: 10%
CutSmart® Buffer: 100%

Diluent Compatibility

Storage Temperature


Storage Conditions

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 min

Methylation Sensitivity

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

Heat Inactivated


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.
  • 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.
  • 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.
  • RNase Activity (2 Hour Digestion):
    The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

Supporting Documents

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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]
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.


  1. 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).
  2. Use of excess enzyme inhibits cleavage. Optimization of the amount of enzyme needed for complete digestion may be required for each substrate DNA.
  3. 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.
  4. 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).


  1. Zheng, Y. et al. (2010). Nucl. Acids Res. doi:10, 1093/nar/gkq327.
  2. U.S. Publication No. 2010-0167942 Unpublished observation
  3. 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.
  1. How has FspEI enzyme been used for epigenetic studies?
  2. How can I minimize off target or star activity?
  3. Should I add more enzyme to get more cutting?
  1. Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using FspEI