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EpiMark® Methylated DNA Enrichment Kit

Catalog # Concentration Size List Price Quantity Your Price
E2600S 25 reactions $835.00
$751.50
Catalog # Size List Price Your Price
E2600S 25 reactions $835.00
$751.50
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
This kit offers reagent for selectively enriching double-stranded methyl-CpG DNA from fragmented genomic DNA.
  • The methyl-CpG binding domain of human MBD2 protein fused to the Fc-tail of human IgG1 (MBD2-Fc) provides high-affinity binding and greater sensitivity
  • Easy-to-use protocol yields enriched fractions in less than 2 hours
  • Enriched methylated DNA fractions can be easily ligated to double-stranded adaptors for Next Generation Sequencing
  • Each kit contains sufficient reagents for the enrichment of methylated DNA from up to 100 μg of fragmented input DNA
The EpiMark® Methylated DNA Enrichment Kit will selectively bind and enrich double-stranded methyl-CpG DNA from fragmented genomic DNA with as little as 5 ng of input DNA.

Methylated DNA is isolated from fragmented genomic DNA (5 ng–25 μg) by binding to the methyl-CpG binding domain of human MBD2 protein fused to the Fc tail of human IgG1 (MBD2-Fc), which is coupled to paramagnetic hydrophilic protein A beads (MBC2-Fc/Protein A Magnetic Bead). Two Fc domains can be bound to one site on protein A with high affinity (Kd=10-7). As the Fc fragment is a dimer, four MBD2 domains are exposed to the solvent per molecule of protein A, increasing the relative equilibrium constant 100-fold. This stable complex will selectively bind double-stranded methylated CpG containing DNA. After simple wash steps followed by magnetic capture, the enriched DNA sample is easily eluted in a small volume of nuclease-free water by incubation at 65°C. The sample is immediately ready for downstream analysis by a variety of methods including:
  • Endpoint and real-time PCR assays
  • Bisulfite conversion followed by DNA amplification
  • Cloning and sequencing
  • Direct sequencing
  • Library preparation for high-throughput sequencing
  • Labeling for DNA microarray analysis
  • Methylation-sensitive restriction enzyme-based assays

MBD-Fc and MCIp were originally developed by Michael Rehli at the University of Regensburg to improve the sensitivity and specificity of conventional CpG binding techniques.


Kit Components: 

Each kit contains sufficient reagents for the enrichment of methylated DNA from up to 100 μg of fragmented input DNA. If starting with 5 ng to 10 μg of input DNA per experiment, the kit provides sufficient reagents for 25 reactions. Store at 4°C. For long-term storage > 6 months, MBD2-Fc protein should be stored at -20°C.

 

Figure 1. Enrichment workflow

 



Figure 2: Endpoint PCR of a methylated locus (LINE) that has been purified using the EpiMark methylated DNA Enrichment Kit

 

Analyzed fractions are described above gel.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Bind/Wash Reaction Buffer (5X) B1032AVIAL 4 2 x 25 ml 5 X
  NaCl High-Salt Elution Buffer (2M) B1033AVIAL 4 1 x 15 ml
  Fragmented HeLa DNA N0484AVIAL 4 1 x 0.02 ml 100 µg/ml
  LINE Primers for Methylated Controls N0485AVIAL 4 1 x 0.02 ml 100 µM
  RPL30 Primers for Non-methylated Controls N0486AVIAL 4 1 x 0.02 ml 100 µM
  MirA Primers for Input Control N0487AVIAL 4 1 x 0.02 ml 100 µM
  MBD2a-Fc Protein M0358AVIAL -20 1 x 0.025 ml 2 mg/ml
  Protein A Magnetic Beads S1426AVIAL 4 1 x 0.25 ml
Features

  • High-affinity binding provides greater sensitivity.
  • Elution in a small volume simplifies downstream applications.
  • Easy-to-use protocol yields enriched fractions in less than 2 hours.
  • Enriched methylated DNA fractions can be easily ligated to double-stranded adaptors for Next Generation Sequencing.
  • Highly pure product from a wide range of input DNA concentrations.

Properties & Usage

Materials Required but not Supplied


50 bp DNA Ladder (NEB #N3236) 
6-tube Magnetic Separation Rack (NEB #S1506) 
Taq DNA Polymerase with Standard Taq Buffer (NEB #M0273) 
Deoxynucleotide Solution Mix (NEB #N0447) 
Nuclease-free Water

Companion Products
References
  • Cross, S.H., Charlton, J.A., Nan, X., and Bird, A.P. (1994). Nat. Genet. . 6, 236-244.
  • Fraga, M.F. et al. (2003). Nucleic Acids Res.. 31, 1765-1774.
  • Gebhard, C. et al. (2006). Nucleic Acids Res. . 34, e82.
  • Gebhard, C. et al. (2006). Cancer Research . 66, 6118-6128.
  • Jacinto, F.V., Ballestar, E. and Esteller, M. (2008). Biotechniques . 44, 35, 37, 39 passim.
  • Lister, R. et al. (2008). Cell. 133, 523-536.
  • Rauch, T. and Pfeifer, G.P. (2005). Lab Invest.. 85, 1172-1180.
  • Schilling, E. and Rehli, M. (2007). Genomics . 90, 314-323.
  • Weber, M. et al. (2005). Nat. Genet. . 37, 853-862.
Citations
Additional Citations
  • Wippermann A, Klausing S, Rupp O, Albaum SP, Büntemeyer H, Noll T, Hoffrogge R (2014) Establishment of a CpG island microarray for analyses of genome-wide DNA methylation in Chinese hamster ovary cells Appl Microbiol Biotechnol 98(2), 579-89.PubMedID: 24146078, DOI: 10.1007/s00253-013-5282-2
  • McGaughey DM, Abaan HO, Miller RM, Kropp PA, Brody LC (2014) Genomics of CpG methylation in developing and developed zebrafish G3 (Bethesda) 4(5), 861-9.PubMedID: 24657902, DOI: 10.1534/g3.113.009514
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
Legal And Disclaimer

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.MBD-Fc and MCIp was developed by Michael Rehli at the University of Regensburg to improve the sensitivity and specificity of conventional CpG binding techniques.

Limited Use Label License: This product and its use is the subject of one or more issued and/or pending U.S. and foreign patent applications owned by SEQUENOM, Inc. The purchase of this product from NEB or its authorized distributors conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity). The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications directed to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use this product for the following COMMERCIAL PURPOSES: (1) use of the product or its components in manufacturing; (2) use of the product or its components for therapeutic, diagnostic or prophylactic purposes in humans or animals; or (3) resale of the product or its components, whether or not such product or its components are resold for use in research.

ACCEPTANCE OF THE FOREGOING LIMITED USE PROVISIONS: If the purchaser is not willing to accept the limitations of this limited use statement, NEB is willing to accept return of the product with a full refund. For information on purchasing a license to use this product for purposes other than those permitted above, contact the NEB Licensing Department at busdev@neb.com.

The MBD-Fc technology is licensed exclusively to New England Biolabs from Sequenom, Inc.

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