ChIP- any experiences in using restriction enzymes instead of sonication? - (Feb/06/2007 )

Hi,

I was wondering if any forum members have tried to use restriction enzymes for shearing the DNA instead of sonication? Perhaps someone has done both sonication and enzyme cutting and gotten good results? Any help, or suggestions are appreciated.

Thanks,
Mads

hi mads,

there are some papers that have used micrococcal nuclease digestion for chromatin fragmentation. I don't think restrcition enzymes would work very well on chromatin as there are proteins/histones bound to the DNA that would not make restriction digestion very efficient if at all.

Nick

Hi Nick,

Thanks for the answer. I have seen an article, Lee Kang et al. 2002, where they combine ChIP and DNA footprinting, and they use PstI for digestion of the protein bound DNA, BUT the use >500U to do it. My problem is that I need to be sure that I get rid of a region nearby, so I have to "split" the DNA more precisely, than I am able to with sonication. I havent been able to come up with any good ideas to do this, which is why I'll try this restriction enzyme stuff.

Mads

Hi Mads, I have only done a little ChIP before but then I used 200U MNase (in combination with some light sonication) to cut my DNA and it worked quit good. But you have to be careful so that the area you are interested in are not totaly chewed up. In my case the nuclesomes were mapped, and I also checked the cutting frequency in closed chromatin (silenced gene) compaired to when the gene was in an activly transcribing state. In my studie I was interested in looking at Histone modifications so I don´t know if this will be of any help for you.

Good luck Matilda

Anyone please tell me with what would you reconstitute the enzyme micrococcal nuclease(water or 0.1%BSA or any other buffer)? and to what concentration would you reconstitute it to have maximum activity.