CHIP questions - proteinase K digestion and DNA shearing - (Mar/15/2006 )

Greetings,

I'm a newbie to both ChIP and this forum, so please bear with me. I've been going through a lot of ChIP protocols to find one suitable for me (read: SHORT) and they all have a proteinase K step after de-crosslinking. Since the DNA is going to be extracted using phenol/chloroform (or alternatively a column), why do we need to predigest the protein wth proteinase k? Couldn't I just go straight to the P/C step after de-crosslinking?

Also, has anyone tried using a large guage (narrow orifice) needle to shear DNA rather than sonicating? I've had some experience with sonication before and I must say I hate the lack of reproducibility.

Any suggestions are welcome.

Thanks.

Reverse crosslinking is carried out in the presence of proteinase k at 65C. I think proteinase k is necessay and mere heat cannot release DNA from proteins.

I seriously doubt you can achieve adequate fragmentation (below 1kb) by needle shearing.

Krakus

The reason people use a proteinase K digestion prior to PCI extraction is probably because that's how it was described in the Chromatin issue of Methods in Enzymology (304). The authors used it to dissolve chromatin aggregates that formed after cross-link reversal. I've heard folks say that it's unnecessary, and in theory, it really should be. But as they say, when it ain't broke, don't fix it.

An alternative to sonication is micrococcal DNase treatment. You'll have to optimize incubation times for each sample type, but it's really the only alternative to sonicating.

Best,
Statler