Chromatin IP question

[Dr. Chiquita]

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Hey peeps. I was wondering if you guys can help me out with my ChIP protocol. This is out of mammalian cells (MCF-7 to be exact). My sonication step basically chops down DNA too much (100~150 bp) instead of 300~700 bp of where I want to be. I tried many different sonication conditions and no matter what, I can't get a size bigger than ~150 bp.

Help.

 

[NGN47]

I have used a Branson Digital Sonifier at 25% power four times each for 30 seconds. This works for HeLa cells. Which sonifier are you using?

Chromatin IP experiments are generally difficult to get to work, so do not give up hope! Good Luck.

 

[spinman]

Consider enzymatic digestion of the DNA as an alternative (MNase, DNase) - slightly better control but a bit more time consuming.

 

[Dr. Chiquita]

I have used a Branson Digital Sonifier at 25% power four times each for 30 seconds. This works for HeLa cells. Which sonifier are you using?

Chromatin IP experiments are generally difficult to get to work, so do not give up hope! Good Luck.

I can't find the name of this sonicator. Ultrasonic by Heat Systems? At least I am somewhat comforted by the fact that it's a tricky protocol for everybody, not just me. Thanks. 🙂

 

[Dr. Chiquita]

Consider enzymatic digestion of the DNA as an alternative (MNase, DNase) - slightly better control but a bit more time consuming.

Wouldn't this introduce non-randomness of these enzymes for certain sites on DNA for digestion? Also, wondering why using these enzymes is more time consuming than sonication? Are you talking about incubation time? Sorry for so many questions. I am thinking about buying MNase.

By the way, I'm doing X-ChIP, not N-ChIP.

Thanks for all your help. 🙂

 

[DrTacoElf]

ah mcf-7. i grow those breast cancer cells sometimes.