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We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios with your chromatin analysis experiments.
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First make sure that the PCR condition is fully optimized and check the primer design. Then try increasing the amount of template DNA added to the PCR reaction. Finally, evaluate sonication of nuclei by microscope to ensure complete lysis.
This is likely to be caused by insufficient chromatin amount in the IP reaction or insufficient antibody incubation time. We also recommend optimizing the crosslinking condition, and monitoring sonication of nuclei by microscope to ensure complete lysis.
The most possible cause is the antibody does not function in IP. Not all antibodies used for western blotting will work well in ChIP. You need to verify the antibody is qualified for ChIP or IP applications. And try adding more antibody to the IP reaction and more DNA template to the PCR.
There might be excess chromatin or antibody added to the IP, or insufficient amount of DNA template added into the PCR reaction. Also your PCR conditions might need optimizing. Try decreasing the number of amplification cycles in PCR. Finally, it is ideal to have duplicate or triplicate runs for each IP to identify any issues, like human or product errors.
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