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 μ g) 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
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.
Required Materials Not Included:
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
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