I am planning to perform ChIP analysis using MNase based method from Pierce ChIP kit. The manual that comes with the kit confused me. It says we should perform sonication (20-second pulses at 3 watts power using Sonicator 3000) to break the nuclear membrane and release chromatin after MNase digestion. Doesn't sonication further shear the DNA? I didn't see any mononucleosom or dinucleosome bands on my gel so I supposed the sonication step did something funny. Would anyone recommend an alternate way to break nuclear membrane? Thanks!
Hello.
Typically, the nucleosomes have a good grip with DNA and survive sonication even in non-crosslinking conditions. You may try to break the nuclear membrane with detergents. I should mention that MNase only treatment could introduce some bias towards underrepresentation of heterochromatin, because euchromatin is much easier to acess.
Unfortunately, since the kit buffer recipe is typically not published, I cannot give a suggestion how to tweak your experiment.
With best regards,
Anatoliy
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