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Temperature is critical. Perform cell lysis at 4°C. Keep the samples ice-cold and use ice-cold buffers.
When using paraformaldehyde, ensure that it is freshly prepared (final concentration of 1%–1.5%).
Usually, a 10-minute incubation at room temperature in agitation is sufficient.
Under cross-linking can prevent the dissociation of protein-DNA complexes and can result in poor yield.
Over cross-linking can mask epitope sites crucial for antibody binding, prevent proper chromatin shearing, and inhibit the successful uncross-linking of the complex in subsequent steps.
Make sure the sonicator probe is not in contact with the tube wall.
Increase the number of sonication steps; however try to avoid increasing the time (or the power) of each step as this may overheat the sample and lead to the loss of antigenicity.
Add ice to the sonicator to avoid the sample overheating.
Preferably do not sonicate chromatin to a fragment size of less than 500 bp (perform the size-testing step).
Different cell types may have different optimal DNA fragmentation. Determine appropriate sonication times to get your optimal DNA fragmentation.
Always fully resuspend beads by vortexing before pipetting.
Always store at 4°C and never allow beads to dry out.
Check the subclass of your antibody is compatible with Protein A/G.
Insufficient antibody can result in too little material for successful PCR analysis.
Too much antibody can increase PCR background.
Usually a 15-minute incubation at 95°C is sufficient. However, with some samples, Proteinase K treatment for 4 or more hours at 65°C may be necessary.
Use different washing buffers (low & high salt, LiCl, and TE buffers).
While using a commercial purification column, check the column is completely dry after the wash step as any leftover wash will inhibit elution.
Make sure the elution buffer is placed directly onto the silica membrane and allowed to adsorb for at least 1 minute.