Product Class: Kit

EpiMark® Bisulfite Conversion Kit

This product was discontinued on December 15, 2022. Alternative products are the NEBNext® Enzymatic Methyl-seq Conversion Module (NEB #E7125) and NEBNext® Enzymatic Methyl-seq Kit (NEB #E7120).

  • Catalog # E3318 was discontinued on December 15, 2022

Product Introduction

Bisulfite conversion is the most commonly used technique to date and is the “gold standard” for methylation analysis.

  • Incubation of the target DNA with sodium bisulfite results in conversion of all unmodified cytosines to uracils leaving the modified bases (5-mC or 5-hmC) intact
  • Complete conversion of Cytosine is achieved by alternating cycles of thermal denaturation with incubation reactions
  • This kit provides materials for 48 reactions
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Product Information

Description

Genomic DNA from many organisms has modified nucleotides. In the mammalian genome, the modified base is predominately 5-methylcytosine (5-mC), which is involved in gene expression regulation, including selective inactivation of one X chromosome in females of mammalian species (1,2). Symmetrical CpG DNA methylation is heritable, but also reversible. This change of methylation status results in an enormous number of combinations of epigenetic states that can regulate gene expression. In mammalian cells, DNA methylation mainly occurs in CpG dinucleotides and is carried out by two methyltransferase enzymatic activities, namely, maintenance methylation and de novo methylation. The maintenance methyltransferase, DNMT1, is involved in the DNA methylation after every cellular DNA replication cycle. DNMT3a and DNMT3b are the de novo methyltransferases that are active in early development.

For optimal results, 50 ng–2 μg DNA is recommended.

Kit Components: 
Each EpiMark Bisulfite Conversion Kit contains sufficient reagents to perform the bisulfite conversion reaction of 48 samples. Store at room temperature (15–20°C). However, for long-term storage (longer than 4 weeks) Desulphonation Reaction Buffer concentrate should be stored at 2–8°C. Prepared Bisulfite Mix can be stored at -20°C for up to six months. 
This product is related to the following categories:

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E3318S     25    
      Desulfonation Buffer (concentrate) B1028AVIAL 25 1 x 3 ml Not Applicable
      Binding Buffer B1029AVIAL 25 1 x 30 ml 1 X
      Wash Buffer (concentrate) B1030AVIAL 25 2 x 10 ml Not Applicable
      Elution Buffer B1031AVIAL 25 1 x 4 ml 1 X
      Solubilization Buffer B1027AVIAL 25 2 x 1 ml Not Applicable
      Bisulfite Mix B1026AVIAL 25 6 x 0.5 g Not Applicable

Properties & Usage

Materials Required but not Supplied

Custom design primers
EpiMark Hot Start Taq DNA Polymerase (NEB #M0490) or other hot start DNA polymerase specific for bisulfite converted DNA.
Heat block or water bath (requiring temperatures of 65°C and 92°C)
96–100% molecular biology grade ethanol
PCR Thermal Cycler
0.2 ml strip tubes and caps
1.5 ml reaction tubes
Nuclease-free Water
Pipettors and pipette tips (for minimization of cross contamination, use aerosol
barrier tips)

Method Overview

Many tools can be used to determine the methylaton status of DNA, including antibodies, methyl binding proteins, methylation-dependent restriction enzymes, and most recently next generation sequencing. However, the most commonly used technique to date is sodium bisulfite conversion, the “gold standard” for methylation analysis (3). Incubation of the target DNA with sodium bisulfite results in conversion of all unmodified cytosines to uracils leaving the modified bases (5-mC or 5-hmC) intact. (see Figure 1). The most critical step in methylation analysis using bisulfite conversion is the complete conversion of unmodified cytosines. This is achieved by alternating cycles of thermal denaturation with incubation reactions. The protocol optimized for this kit is simple and gives consistent results.

Figure 1. Overview of Bisulfite Conversion ReactionFigure 1. Overview of Bisulfite Conversion Reaction

Methylated cytosines are protected and remain unchanged, while unmethylated cytosines are deaminated to uracil after treatment with sodium bisulfite.
Figure 2. Performance evaluation of the EpiMark Bisulfite Conversion Kit.Figure 2. Performance evaluation of the EpiMark Bisulfite Conversion Kit.

One μg of genomic DNA was bisulfite treated using the Epimark Bisulfite Conversion Kit, and 2 μl of eluted DNA was analyzed by end-point PCR. 388, 544, and 731 bp amplicons were amplified with primer pairs for bisulfite converted DNA (lanes 1, 3, and 5), or with primer pairs for unconverted DNA (lanes 2, 4, and 6). Epimark Hot Start Taq DNA Polymerase was used for amplification. Lanes 2, 4, and 6 clearly show no amplification product, indicating complete DNA conversion. Marker M is the 2-Log DNA Ladder (NEB #N3200).
Figure 3. Conversion of a methylated DNA fragment.Figure 3. Conversion of a methylated DNA fragment.

50 nanograms of methylated plasmid DNA was bisulfite converted and a 146 bp DNA fragment was amplified using Epimark Hot Start Taq DNA Polymerase (NEB #M0490). Amplicons were cloned, and individual clones were sequenced to determine the methylation status. Sequence aligment of 10 clones is shown to demonstrate conversion of unmethylated cytosine to thymine, while the unconverted methylated cytosine remains intact. The known methylated cytosines at the CpG site shown in red, converted cytosines marked as T, and rest of the sequence is shown as a dashed line.

References

  1. Bestor, T.H., Verdine, G.L. (1994). Curr. Opin. Cell Biol. . 6, 380-389.
  2. Ooi, S.K., O'Donnell A.H., Bestor T.H. (2009). J. Cell Sci. 122, 2787-2791.
  3. Frommer, M., et. al. (1992). Proc. Natl. Acad. Sci. USA. 89, 1827-1831.
  4. Kumaki, Y., Oda, M. and Okano, M. (2008). Nucleic Acids Res. . 36,

Protocols, Manuals & Usage

Protocols

  1. Reagent Preparation Using EpiMark® Bisulfite Conversion Kit (E3318)
  2. End-point PCR Using EpiMark® Bisulfite Conversion Kit (E3318)
  3. Cycling Protocol Using EpiMark™ Bisulfite Conversion Kit (E3318)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. How should I store my bisulfite converted DNA after it is eluted from the column?
  2. Does my sample need to be free of RNA for bisulfite conversion?
  3. No PCR product is detected when I amplify more than 300 bp amplicon.
  4. Do I need extra purification of my mammalian DNA prep in order to use it in a bisulfite conversion reaction?
  5. Which DNA polymerase do you recommend for amplification of converted DNA?
  6. My thermocycler does not allow to set up the reaction volume to 140 μl, what I should do?
  7. What is the shelf life of the reagents supplied?

Quality, Safety & Legal

Quality Assurance Statement

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 DataSheets

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

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.

Licenses

Methylation-specific PCR may be covered by one or more of U.S. Patent Nos. 5,786,146; 6,017,704; 6,200,756 and 6,265,171 and patents based on foreign counterpart applications. No license or rights under these patents to perform methylation-specific PCR is conveyed expressly or by implication to the purchaser of this product. User's of New England Biolabs' products should determine whether they have all the appropriate licenses in place. Further, no warranty is provided that the use of these products will not infringe on the patents referred to above.

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