I am very new to ChIP-qPCR and I have several questions that I hope you can help me answer. Along with the input sample, I have three ChIP samples: IgG, UBF (Positive control), and a TF (my experimental sample) and two primer pairs. I am fortunate to have received a really good protocol for the ChIP portion of these assays, but my confusion comes in with the qPCR setup and subsequent analysis.
My first question: is it necessary to calculate your amount of input? Do you need to quantitate the amount of DNA in the input for your analysis or is okay to simply take 100 ul of input and use 1000 ul of input for each ChIP? In the latter case your input would be 10% of what is used for each ChIP.
My second question: what samples do you need to run for qPCR? For each primer pair, I assume you would run a negative control, 5 dilutions of a sample to make a standard curve to test for efficiency, your input sample, and each of your ChIP samples. Are there other samples that need to be run in addition to these?
Final questions: what are peoples' thoughts on doing samples in duplicate versus triplicate? How small a qPCR reaction can one use to get reproducible results? Is a 10 ul total volume for qPCR too small?
Thank you all! I look forward to your insightful comments.