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  • EpiMark® Methylated DNA Enrichment Kit Troubleshooting Guide

    Observation Possible Cause(s) Solution(s)
    No or poor DNA target detection by PCR-based method in unbound and/or elution fraction(s). DNA is degraded. Take precautions to prevent DNA degradation by maintaining a nuclease-free environment. Increase EDTA concentration of sample to 10 mM.
    Not enough target DNA. Verify target DNA concentration by nanodrop instrument, or other sensitive DNA detection system. Run target DNA on agarose gel to determine quantity, quality and size.
    DNA did not elute from the MBD2a-Fc beads. Raise the temperature of the elution to 98°C, mindful that this will render the sample single-stranded.
    Unable to clone eluted DNA fragments. DNA ends are frayed due to sonication or nebulization, or DNA has been rendered singlestranded. Repair DNA ends using a blunt-end repair kit (e.g., Quick Blunting Kit, NEB #E1201).
    Controls did not work, did not see bands as expected on gel. DNA is degraded. See above for DNA precautions.
    DNA did not elute from
    the MBD2a-Fc beads.
    See above for elution at 98°C.
    PCR did not work. Verify that all of the components have been added to the PCR mix. Lower the annealing temperature of the reaction to 55°C.
    Controls did work, but did not see my gene of interest. DNA target does not contain sufficient amounts of CpG methylation. Raise input DNA concentration to at least 1 µg.
    PCR did not work. Optimize PCR conditions for your target sequence.

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