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Old 01-04-2013, 02:48 PM   #13
Hamid
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Location: San Diego, CA

Join Date: Sep 2009
Posts: 105
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Hi Nfourier,

You are over-fixing your cells. I have yet to find a single mammalian cell that requires more than 5 minutes of cross linking at room temperature.
May I suggest that you follow all the steps of either the Covaris truChIP or Ethan's protocol including the fixation, nuclei isolation, and shearing. Please use methanol-free formaldehyde for fixation.
Most chromatin shearing protocols out today were developed and optimized for use with probe or bath sonicators. These protocols will very likely not work with Covaris AFA. I do understand your reluctance to change your protocol, but since Covaris AFA is a much different technology than probe and bath sonication, you will have to change your sample preparation to match the requirements of AFA.

Thank you

Hamid
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