Hello, I'm doing a Chip-seq experiment and I've noticed that after DNA extraction I have the same amount of DNA in my negative control (IgG) and in the IPs? Do you think it's still worth it to make the library and sequence it?
Thanks
It is absolutely OK. DNA concentrations don't depend on IP efficiency and are almost the same in all samples.
Perhaps you could do a quick check using qPCR targeting a gene known to enrich the immunoprecipitated proteins/histone marks
My IgG controls are usually almost undetectable. I agree if you know or can find a negative and a positive region then Qpcr is the best check.
@Nikolay @Jessica and @ Cheng Dong
I spike-in known amount of DNA into weak/ mild TE buffer and perform IP with known Abs (IgG as control and H3 as target binding Ab) and Protein G magnetic beads. My goal is to optimize the IP protocol and enrich the sample. I do Qubit quantification after DNA isolation but unfortunately both control and target seem to have same DNA concentration. I wonder what is being quantified in case of control, when I elute my DNA in Dnase free water and in principal it should not have any trace of DNA ?!
I suspect the buffers that I am working with. Can anyone suggest me some of their buffer recipes ?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques