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Loni Pickle, Life Technologies R&D Scientist, reviews various approaches for chromatin shearing upstream of chromatin immunoprecipitation (ChIP). ...
Loni Pickle, Life Technologies R&D Scientist, reviews various approaches for chromatin shearing upstream of chromatin immunoprecipitation (ChIP).
Tissue Processing Using Magnetic Beads -- Shearing Optimization
Chromatin shearing is the step in ChIP where I receive the most amounts of questions; so let's spend a couple of minutes reviewing this key step. There are two basic methods to shear your samples during ChIP experiments: Enzymatic digestion and Sonication.
Here's some tips that should be useful for when you are optimizing your shearing:
Keep your samples cold -- keep your samples on ice or in cold water alternating cycles of on and off ice to allow your samples to cool between cycles. This will help maintain your DNA protein interactions. Sonication produces heat, that can disrupt your protein DNA interaction.
For ChIP qPCR, shoot for a fragment size between 200 to 500 bp. For ChIP Seq applications using the SOLiD™ platform shoot for a fragment size range from 100 to 300 bp. If you're using another platform, follow that vendor's recommendations. Be sure to verify your fragment size before moving into your immunoprecipitation step of the ChIP protocol. You want to aim for the least amount of sonication cycles and intensity that gives you the correct size distribution.
As we've covered here there are a lot of factors that can influence your fragment size but I think you'll find it's well worth the effort as you head into your downstream application.
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