Bioruptor - Poor Fragmentation Results

[Kolamite]

Hey,

I'm trying to perform a ChIP-seq experiment for a transcription factor using the Diagenode bioruptor.

I'm getting very poor fragmentation at 30 minutes (3x10 cycles, high power, 30s on, 30s off). The Bioanalyzer reports that ~3% of my DNA is in the 200-600 BP size range.

Other details:
-Crosslinking 1% formaldahyde, 10 minutes @ RT
-~200k cells per tube/200 ul volume
-Decrosslinked before QC

After reading a few threads here, I tried reducing my crosslinking time to 5 minutes and ran a timecourse of 10/20/30 minutes on the bioruptor. I ran the products on an agarose gel with similarly uninspiring results.

The one thing I can see that I'm really doing against protocol is using standard polypropylene tubes instead of the TPX ones. I've ordered them, but someone in the lab has warned me that they help, but don't make a massive difference.

Does anyone have other suggestions I can try?

Edit: I also tested the Bioruptor with the foil test and got a good result, so I think the instrument is ok.

Thanks,

[dalib]

Dear Kolamite,

Chromatin shearing is very much dependent on the type of cell, the amount of cells, the final volume, the buffer composition and the type of plastic (TPX tubes improve efficiency but it is still important to optimize the other parameters).

Diagenode has published a guide to chromatin shearing with the Bioruptor, which could be of use to optimize your experiment. You can download it from here: http://www.diagenode.com/media/docum...r_protocol.pdf
For more specific assistance you can always contact our North American technical team ([email protected]).

Daniel