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ChIP with MNase


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#1 Kab

Kab

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Posted 02 July 2010 - 07:05 AM

Hello all,

I am having major problems with ChIP. I was able to get results a couple of times, but now things have stopped working. I am using an Active Motif enzymatic kit, and can not seem to get Input DNA anymore (along with IPed DNA). I have tried increasing lysis time and amount, decreasing formaldehyde fixation time, different strokes on the homogenizer anywhere from 2X to 300X, switched homogenizers (have used both a dounce with a tight-fitting B pestle and potter-elvehjem).

I am using keratinocytes and growing to 100% confluence (does anyone work with ChIP in 100% confluent cells?) because the receptor I'm studying is activated at it's highest level when the cells are fully confluent.

Our lab has tried sonication previously but have had trouble optimizing, so we switched to enzymatic, aka MNase. Anyone have any suggestions? I'm willing to try anything!

Thanks in advance.




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