1. On page 3 of the bulletin, the tissue prep procedure calls for making the cross-link solution with formaldehyde in cell culture medium but there is no mention of cell culture medium anywhere else and why would this be used instead of an extraction buffer? What else can be used?
Cells should be cross-linked (X-linked) in physiological conditions (hence the culture medium) in order to freeze and preserve the DNA-protein interactions in vivo as they happen. X-linked cells are later swollen in hypotonic solution, homogenized to release nuclei and the chromatin is extracted in nuclei lysis buffer.
3. Is the kit compatible with lysing enzymes if sonication does not work?
Yes, sheared chromatin can also be made by enzymatic (Micrococcal nuclease, MNase) digestion (reagents/protocol not provided in kit). The amount and duration of the MNase treatment will have to be optimized by the user depending on the cell line.
5. I am having trouble with sonication. What is the recommended time for sonication?
Sonication has to be optimized for each cell line and the instrument, we recommend the Diagenode Biodisruptor (water based sonication) for reproducible sonication. A good starting point is 5, 10 and 15 minutes at High “H” setting with 30 seconds “on” and 30 seconds “off” cycle.
Run a gel to check sonication:
6. What is the function of nuclear preparation buffer? When do you add the protease inhibitor?
The nuclear preparation buffer is a hypotonic salt solution that serves to swell the cells and facilitate the release of nuclei during the subsequent homogenization step. The protease inhibitor cocktail should be added just before use.
7. Can this kit be used with plants? Reptilian cells?
Yes, the kit is compatible with sonicated chromatin prepared from plants or reptilian cells provided the user has optimized conditions of cross-linking and preparation of appropriately sized sonicated chromatin.
10. Can the number of cells be increased? What is the maximum and minimum number of cells that can be used with this kit?
The range of cells that can be used with this kit is 0.1-1 million/well/ChIP sample, using more than 1 million cells per well will increase the non-specific binding and reduce the specificity of the ChIP reaction. If highwe yield of ChIP DNA is desired DNA could be pooled from multiple individual ChIP reactions for downstream processing (labeling/ hybridization) or DNA could be amplified using the WGA2 kit for ChIP-chip analysis.