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ChIP assay using heart tissue?

ChIP tissue enzyme digestion

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#1 swchoi

swchoi

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Posted 29 November 2011 - 03:38 PM

Hello, I have never done ChIP assay before, thus my question may be very ignorant..I am planning to do ChIP assay using heart tissue, and immediately found two problems:
1. how to lyse/homogenize heart tissue? it is pretty tough tissue, not well homogenized by glass homogenizer..

2. I bought enzyme-digest based ChIP assay kit(active motif), by thinking enzyme digestion may be easier than sonication, but found it in the protocol that I can't use column to purify DNA but to use chloroform/phenol method and suggested protocol is.....very tricky and time consuming. Is column purification really NO NO in enzyme digested based ChIP?

3. any simplified ChIP method to suggest? Even with kit, it takes forever to do so...I have quite a bit of samples thus the simpler/quicker the better.

4. I am planning to perform qPCR at the end to quantitate. Then what is the criteria to design the primer for ChIP-qPCR? Usually putative response element is <20bp, then PCR should cover only that region? Or make it 100-200bp around it? Any online tool to do so?

Thank you so much for your advice in advance.

:-)

Sung





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