I have performed the ChIP and quantify the samples using Qubit Flurometer 2.0. Here are the results:
Untagged control :2.72ng/mL
Tagged sample: 3.17ng/mL
I think the untagged strain should have much lower DNA concentration compared to those with tagged. Is this due to the non-specific binding?
What can I do to troubleshoot it?
Firstly the difference between your tagged and untagged samples are not that large. Second even if you see a larger difference that still cant tell much about your ChIP results. Its the enrichment score at a particular loci that is more important than the over all DNA amount. Many a times IgG control samples pull down large amount of DNA than the target Ab samples, but we see no net enrichment at any particular loci, rather a generalized background, whereas target Ab ChIP show a more focused loci specific enrichment. There a several methods to reduce nonspecific pull down
1. Lesser amount of Ab to be used
2. Preclearing of chromatin with Agarose /Dynal beads
3. Blocking of beads with BSA or other blockers
4. Extensive and stringent wash with higher salt buffers
5. Ensure that reverse cross-linking overnight is complete
6. Complete removal of RNAse, protenase K and purify good quality DNA
Hope this helps !
All the best
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