Hello,

I'm currently working out conditions for a 4C (circular chromatin conformation capture) experiment, and most of the enzymes that generate acceptable viewpoints can only be heat-inactivated at 80 degrees. This treatment must cause the DNA-bound proteins to denature, because my sample becomes cloudy. Inactivation by EDTA won't work, because the next step is ligation. I found a protocol (van de Werken et al. 2012) that recommends heat inactivation "if necessary" at 65 degrees, and their example enzyme is HindIII, which inactivates at 80 degrees.

Does denaturing the DNA-bound proteins cause problems in 4C? Are there situations in which the RE does not need to be inactivated? Does 20 minutes at 65 degrees sufficiently inactivate an RE with a higher inactivation temperature? Do you have any other suggestions for inactivating the REs?

Thanks!