FspEI

Product Information

• Reagents Supplied

  The following reagents are supplied with this product:

  NEB https://www.neb.com/en/#	Component Name	Component https://www.neb.com/en/#	Stored at (°C)	Amount	Concentration
  R0662S     -20       FspEI R0662SVIAL -20 1 x 0.04 ml 5,000 units/ml   rCutSmart™ Buffer B6004SVIAL -20 1 x 1.25 ml 10 X   Enzyme Activator Solution S0538SVIAL -20 1 x 0.1 ml 15 µM

• 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

  • Diluent B

  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
  

• Related Products

  Companion Products

  • Monarch® DNA Gel Extraction Kit
  • Monarch® PCR & DNA Cleanup Kit (5 μg)

  Materials Sold Separately

  • NEBuffer 4

• Product Notes

  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).
  5. Not sensitive to CpG, dcm, or dam methylation.
  6. star activity may result from extended digestion, high enzyme concentration or a glycerol concentration of >5%.

• References

  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.

Protocols, Manuals & Usage

• Protocols

  1. Restriction Digest Protocol
  2. Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI, FspEI or LpnPI

• Usage Guidelines

  • Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
  • Activity of Restriction Enzymes in PCR Buffers
  • Cleavage Close to the End of DNA Fragments
  • Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
  • Double Digests
  • Heat Inactivation
  • NEBuffer Activity/Performance Chart with Restriction Enzymes
  • Optimizing Restriction Endonuclease Reactions
  • Restriction Endonucleases - Survival in a Reaction
  • Restriction Enzyme Diluent Buffer Compatibility
  • Restriction Enzyme Tips
  • Single Letter Codes
  • Star Activity
  • Traditional Cloning Quick Guide

Tools & Resources

• Selection Charts

  • Alphabetized List of Recognition Sequences
  • Comparison of Methods for DNA Methylation Analysis
  • Compatible Cohesive Ends and Generation of New Restriction Sites
  • Dam-Dcm and CpG Methylation
  • Enzymes with Nonpalindromic Sequences
  • Frequencies of Restriction Sites
  • Isoelectric Points (pI) for Restriction Enzymes
  • Isoschizomers
  • NEB Diluent and Buffer Table
  • Restriction Enzymes for Epigenetics Selection Chart
  • Why Choose Recombinant Enzymes?

• Web Tools

  • Competitor Cross-Reference Tool
  • DNA Sequences and Maps Tool

  • Double Digest Finder
  • Enzyme Finder
  • NEBcutterâ„¢ v3.0
  • NEBioCalculator®
  • REBASE®

FAQs & Troubleshooting

• FAQs

  1. How can I minimize off target or star activity?
  2. Should I add more enzyme to get more cutting?
  3. Is this enzyme sensitive to dam, dcm or mammalian CpG methylation?
  4. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

• Troubleshooting

  • Restriction Enzyme Troubleshooting Guide

Citations & Technical Literature

• Citations

  Product Citation Tool

Quality, Safety & Legal

• Quality Control Assays

  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 & Change Notifications

  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]

  • R0662S_L_v2

• 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]

  • R0662S_L_v1_0011211
  • R0662S_L_v1_0011303
  • R0662S_L_v1_0011310
  • R0662S_L_v1_0011502
  • R0662S_L_v1_0011601
  • R0662S_L_v2_0011605
  • R0662S_L_v1_0011605
  • R0662S_L_v2_0041612
  • R0662S_L_v2_0041708
  • R0662S_L_v2_0041710
  • R0662S_L_v2_0041804
  • R0662S_v2_10025342
  • R0662S_v2_10038404
  • R0662S_v2_10040506
  • R0662S_v2_10060447
  • R0662S_v2_10072892
  • R0662S_v2_10079662
  • R0662S_v2_10090232
  • R0662S_v2_10100421
  • R0662S_v2_10108701
  • R0662S_v2_10132917
  • R0662S_v2_10152970

• Safety Data Sheets

  The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

  • FspEI
  • rCutSmart™ Buffer
  • Enzyme Activator Solution

• Legal and Disclaimers

  Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
  
  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.
  
  New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.