Hello, my name is Pablo, and I am currently optimizing a Chromatin Immunoprecipitation (ChIP) protocol. I am encountering an issue with DNA fragmentation and would appreciate your insight.
Here is a summary of my process:
Nuclei Isolation: I use a lysis buffer containing 0.5% NP-40 to isolate nuclei from 8 million cells.
Nuclear Lysis and Chromatin Preparation: The nuclei are then resuspended and lysed in 700 µL of a second lysis buffer containing 1% SDS to release the chromatin.
Sonication: I take 100 µL aliquots of this lysate and sonicate them using a Diagenode's Bioruptor. The settings are 20 cycles of 30 seconds "ON" and 30 seconds "OFF" (at 4°C).
Analysis: Following sonication, I purify the DNA and run it on a 1% agarose gel to check the fragment size.
The Problem: Despite this process, I do not see the expected smear of fragmented DNA on the gel. The DNA appears to be largely intact or high molecular weight.
What could be going wrong? Have you had a similar experience? Any advice would be greatly appreciated.
Best regards,
Pablo
Hello Pablo,
The most likely reason for not seeing fragmented DNA after sonication could be insufficient sonication energy or time. The settings that you've used might not be enough to shear the DNA to the desired fragment sizes, especially if the DNA is tightly packed within the chromatin or if the sonicator settings need to be optimized for your specific cell type and sonicator model. You may try to increase the number of sonication cycles. Double the cycles to 40. You should also optimize pulse duration. Experiment with different pulse durations. While 30s ON/30s OFF is a common starting point, try shorter or longer pulses which might turn out to be more effective. Ensure the sample is kept cool (ideally 4°C) during sonication to prevent DNA degradation from heat.
Consider using a sonication efficiency kit (it may be available from Diagenode or other vendors) to verify that the sonicator is delivering the intended energy to the sample. If at all you fail, then there are other sonication methods, like probe sonication which offers focused energy for faster and more effective results.
Best,
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