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Native ChIP and sonication with bioruptor, is it possible?


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#1 mosso2000

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Posted 14 May 2009 - 05:52 AM

Hi all, this is my first post on this board, and I was wondering if I could use the bioruptor to shear the chromatin under native non-crosslinked conditions? I use it routinely for crosslinked samples and have an efficient protocol; this would be to perform a histone ChIP.
Thanks in advance for your help!

#2 Stefan

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Posted 20 May 2009 - 08:50 PM

Hi all, this is my first post on this board, and I was wondering if I could use the bioruptor to shear the chromatin under native non-crosslinked conditions? I use it routinely for crosslinked samples and have an efficient protocol; this would be to perform a histone ChIP.
Thanks in advance for your help!



Hi there,
I also use a Bioruptor and for my first test sonication I used native non-crosslinked chromatin and got 500 bp fragments after 5 to 10 min of sonication (30sec ON/30sec OFF). Then I used crosslinked chromatin and figured out quickly that it takes much longer to get fragments under 1000 bp.
So I would say that you can use the Bioruptor for sonication of native non-crosslinked DNA however be aware that the total sonication time should be much shorter...

I hope that helps a bit. I also have a question as I am a fairly new Bioruptor user:
I need to get my crosslinked chromatin to a size of under 500 bp, ideally 200 to 400 bp for ChIP-SEQ. I am using stomach tissue and disaggregate the tissue with a Medimachine (BD) to get a single cell suspension. After nuclei extraction and lysis I start sonicating. I could not figure out yet how long my total sonication time needs to be to get fragments under 500 bp. I know that after 25 min all my fragments are under 1000 bp, but when sonicating for 30 and 35 min I have the same results. So I somehow couldn't get it smaller than 1000 bp yet. What do you think? Just sonicate longer, like 1 h total time or even longer? Do you have any experience with that? What is your optimal fragment size and how long do you need to sonicate to get it?

I hope I could help and hope you also have some answes for me.

Cheers,
Stefan

#3 HP1b

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Posted 24 June 2009 - 02:18 AM

I hope that helps a bit. I also have a question as I am a fairly new Bioruptor user:
I need to get my crosslinked chromatin to a size of under 500 bp, ideally 200 to 400 bp for ChIP-SEQ. I am using stomach tissue and disaggregate the tissue with a Medimachine (BD) to get a single cell suspension. After nuclei extraction and lysis I start sonicating. I could not figure out yet how long my total sonication time needs to be to get fragments under 500 bp. I know that after 25 min all my fragments are under 1000 bp, but when sonicating for 30 and 35 min I have the same results. So I somehow couldn't get it smaller than 1000 bp yet. What do you think? Just sonicate longer, like 1 h total time or even longer? Do you have any experience with that? What is your optimal fragment size and how long do you need to sonicate to get it?

I hope I could help and hope you also have some answes for me.

Cheers,
Stefan


What´t your cross-linker? Formaldheyde, I guess. What´s your final concentration? You could try either to use a bit lower concentration of crosslinker or to reduce the crosslinking time.
I would not reccomend to increase the sonication time: the shorter time you could use to get a well sheared chromatin, the better.

I hope it might help.




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