ChIP sonication problem: too wide range of smear - too wide range of smear (Jun/05/2006 )

Hi All,
I am just starting to do ChIP. My problem with the sonication is that when I tested the sonicated DNA on 1% gel, I saw a smear ranging from 200 bp to 4kb (even after I treated with RNase A). There were no bands, though, just smear. Any suggestion will be appreciated. Thanks!!!
--Sure

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I don't think the smear is RNA, but simly genomic DNA that you have successfully sheared...

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Thanks!
I don't think the RNA is a problem, either. However, my question is, how to make the smear range less wide, like within 500bp-1KB. This is important for my experiment, because I will use the ChIP products on microarray.
Thanks again!!

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Hello,

To get DNA in the 1kb-500 bp range I used to sonicate 5 times 15s at 5 level Power on a XL Misonix. Between each sonication I put my samples back into ice.

If it is not enough you can maybe try to increase the number of sonication.

Hope it helps.

Vero

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hi sure,

not too sure which sonication machine you are using, but with a branson sonifier with a microtip attachment, at 30% duty cycle, 10 15 second pulses usually does the trick for me, i work with mammalian cells. a lower setting on your sonication machine may do the trick.

Nick

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Hello,

I am going to be starting ChIP analysis soon, could someone with more experience provide a gel image shot of what good shearing looks like. I am unsure how discrete a band I should see around 500bp.

Thanks

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here it is http://www.protocol-online.org/forums/inde...showtopic=12700

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I think the wide range of smear is indicative of inadequate sonication. If you sonicate more, you will see a smear from 1 kb to 200 bp with 500 bp being the most intense area.

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Thanks very much for the prompt reply PCRman. So lane 4 would be a good sonication.

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A potential cause is that the volume of your samples is too big so that the DNA is not evenly sonicated.

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