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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