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
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 5 X NaCl High-Salt Elution Buffer (2M) 4 500 mM MBD2a-Fc Protein 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 250 μl
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