Hello,
I have started doing histone acetylation study. First we are looking into 1 gene in our 10 samples.
As i dont have that much experience here, i want to make sure i am doing it properly, so need expert's feedback.
I am using millipore kit, 50 microlt of each IP has 1 million of cell equivalent, saving 1% for input. In RT, my total reaction volume is 25 microlt and i am using 12.5 microlt of SYBR along with total 100 nm primer (FP+ RP). I tried different DNA dilutions, like used 2 microlt and then 50 fold, 100 fold, 200 fold dilutions. For all of the these dissociation curves look good. 2.5 microlt of H3K14 acetylated antibody(Millipore) / IP i am using (as this is not purified, no concentration is written).
the Cts i got for one of my samples are : Input 26, IP 25.5, IgG 34.
Do you think, i am doing it properly and the results i am getting are ok? Do you think i need to optimize any other things like antibody amount or something else?
Another question, as i have 10 samples, do i need to do igG for each of the samples?
Thanks,
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