Epigenetics

ChIP Troubleshooting Questions

 

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.

2. What if there are less than 8 samples, can the strip wells be used partially?
Yes, one can use as few wells as one desires (depending on the number of samples).

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.

4. What is the GAPDH primers efficiency for doing qPCR?
The GAPDH primers efficiency for doing qPCR is 95 %.

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:

  1. Use 10 µl sample and add 40 µl H2O
  2. Reverse cross-link by adding 2 µl of 5 M NaCl (Final concentration 0.2 M NaCl)
  3. Boil for 15 minutes
  4. After returning to room temperature, add 1 µl of 10 mg/ml RNase A at 37 °C for 10 mins
  5. Clean and purify DNA with Sigma’s GenElute PCR purification kit
  6. Load 1 and 4 µl of sonicated DNA on gel and determine size of smear
  7. The sonication condition that gives a smear of DNA sizes from 200 bp to 1 kb with a peak around 500 bp (2-3 nucleosomes) should be used for ChIP reactions.

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.

8. Do we have a protocol for enzymatic digestion of the DNA as opposed to sonication?
No, we have not optimized enzymatic digestion of chromatin but a Pubmed search will reveal these protocols.

9. How do I test if the DNA is sheared since it is not noted anywhere on the protocol?
Run a gel to check sonication:

  1. Use 10 µl sample and add 40 µl H2O
  2. Reverse cross-link by adding 2 µl of 5 M NaCl (Final concentration 0.2 M NaCl)
  3. Boil for 15 minutes
  4. After returning to room temperature, add 1 µl of 10 mg/ml RNase A at 37 °C for 10 mins
  5. Clean and purify DNA with Sigma’s GenElute PCR purification kit
  6. Load 1 and 4 µl of sonicated DNA on gel and determine size of smear
  7. The sonication condition that gives a smear of DNA sizes from 200 bp to 1 kb with a peak around 500 bp (2-3 nucleosomes) should be used for ChIP reactions.

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 WGA 2 kit for ChIP-chip analysis.