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Whole tissue starting product - not cultured cells


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#1 BW.

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Posted 19 July 2009 - 04:42 PM

I was wondering if anyone has been doing ChIP on whole tissue pieces of about 50mg? I have done this previously about 18 months ago with reasonable success. My main query is about the fixation step and fragmentation. Previously I was using 1% formaldehyde fixation with a quenching step, followed by sonication. All done on the whole piece of tissue. The sonication broke the tissue up, then pellet cell debris and continue as standard chip protocol.

Is this sufficient for penetration into the tissue?

Would appreciate any feedback.
Thanks

#2 KPDE

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Posted 19 July 2009 - 10:15 PM

I was wondering if anyone has been doing ChIP on whole tissue pieces of about 50mg? I have done this previously about 18 months ago with reasonable success. My main query is about the fixation step and fragmentation. Previously I was using 1% formaldehyde fixation with a quenching step, followed by sonication. All done on the whole piece of tissue. The sonication broke the tissue up, then pellet cell debris and continue as standard chip protocol.

Is this sufficient for penetration into the tissue?

Would appreciate any feedback.
Thanks


I usually mince the tissue in the formaldehyde at the beginning of the crosslinking. I usually use about 0.1cc sized tissue fragment and mince with forceps till I get very small fragments. Formaldehyde is a very small molecule but I don't know how much it can penetrate in 10 minutes.

#3 epi123

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Posted 24 July 2009 - 07:04 AM

along these lines...does anyone have a detailed tissue chip protocol they can share?

thanks for any help




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