I am going to run some ChIP assays (qPCR based, not ChIP-seq) and am just wondering how important it is to include RNAse and Proteinase K treatment steps after elution from the beads and before the overnight reverse cross linking step. I have heard some say it is not needed but many protocols include it. Does the Prot-K help with the reverse cross links or protect the DNA by degrading nucleases? Not sure why RNA removal is important, as surely this will not interfere with the final PCR.
Any ideas?
I never used RNAse for my ChIPs and they were fine, not really sure what the point of it would be. I always used proteinase K, but never actually verified that it was necessary.
Also, for the reverse cross-linking step, I didn't do O/N, I just used incubated at 95C for 10 min and it seemed to work fine. I think I picked up that idea from reading through the directions in the Millipore kit; can chop off one day of what is already a long protocol.
Thanks. Reading around this, apparently RNAase is needed if you are using column purification as the RNA competes with the DNA for binding but it is not needed for phenol chloro cleanup. Also, some say that ProtK helps with the reverse cross linking.
Thanks for the 95oC 10 min. Worth a try.
Ah, interesting; well maybe it depends on your lysis protocol up front since I used column purification at the end. I always removed the cytosolic fraction and just did a nuclear lysis; perhaps in that way there is much less RNA to compete with in the first place.
Are you sure that a 10min 95deg step is sufficient for reverse cross-linking Justin? That sounds like a ProtK denaturation step to me. All the protocols I've seen use at least 2hr at 65deg for the reverse cross-link step.
I could be wrong here, but in my protocol I had a 2 hr 62C step w/ the prot. K followed by the 95C for 10 min in an elution buffer without NaCl (1% SDS, 0.1M NaHCO3). In the activMotif ChIP kit protocol they suggest a 95C for 15 min prior to the prot. K step, and Novus seems to suggest this is to reverse crosslinking too (http://www.novusbio.com/support/support-by-application/chromatin-immunoprecipitation/protocol.html)....
But perhaps it's the combination of the 2 hr 62C (+ prot. K) step and the 95C step? And perhaps it may also depend on the composition of the buffer? (There's a brief discussion on this here as well: http://www.protocol-online.org/biology-forums-2/posts/13560.html). When setting up my ChIP protocol I didn't run through all the different iterations, just found a combination that worked and stuck with it.
Hi,
I do not use RNAse with my ChIP assays (no columns). But, might help.
To revert cross-linking I add 5 M NaCl and incubate at 65 oC for at least 4 h.
I also believe that the proteinase K treatment is important with the reverse cross linking, I always use it (1h at 45oC).
Good luck!
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