EpiMark® Methylated DNA Enrichment Kit
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.
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.
The following reagents are supplied with this product:
Store at (°C) Concentration Bind/Wash Reaction Buffer (5X) 4 5X NaCl High-Salt Elution Buffer (2M) 4 500 mM MBD2a-Fc Protein -20 2 mg/ml Fragmented HeLa DNA 4 100 µg/ml LINE Primers for Methylated Controls 4 100 µM RPL30 Primers for Non-methylated Controls 4 100 µM MirA Primers for Input Control 4 100 µM Protein A Magnetic Beads 4 250 µl
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