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ChIP sonication fragments too small

ChIP sonication fragments too small - ChIP Chromatin Immunoprecipitation Forum

ChIP sonication fragments too small - Chromatin Immunoprecipitation Forum. Discuss, chat about, and post questions about the ChIP assay, in the ChIP forum. Includes troubleshooting, protocols, buffer preparation, and more.


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Old 06-01-2011, 10:08 PM
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Default ChIP sonication fragments too small



I haven't seen this problem reported before, and was wondering if anyone had any thoughts or solutions.

We are sonicating formaldehyde cross-linked DNA for ChIP using a Misonix S-4000 sonicator. After running the fragments on a gel, we see a smear up to about 3 kb, and bright band of fragments around 100-200 bp (about mononucleosomal length). If we continue to sonicate, eventually the smear will disappear, but all fragments will be 100-200 bp. We never see the enrichment of fragments from ~200-500 bp that we are aiming for. The fragments are always too small.

We've tried cross-linking from 2-30 minutes, using 0.5-2% formaldehyde, different cell densities, a range of amplitudes and times for sonication. We de-crosslink at 65C overnight, and treat with proteinase K and RNAse A. The samples are from asynchronously growing Hek-293 cells.

Has anyone else seen this, or have any experience with getting too much enrichment on short fragments?

Thanks,
Jeff
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Old 02-14-2012, 07:55 PM
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Default Re: ChIP sonication fragments too small

how about trying to sonicate for shorter time, and resting in between? vortexing and mixing in between might allow better mix of sampels whiel sonicating too

Last edited by admin; 02-15-2012 at 04:00 AM.
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Old 02-14-2012, 07:56 PM
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Default Re: ChIP sonication fragments too small

i'm not sure about the sonicator model you use. how does it work? is it direct or by sound waves?
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