Dear All,
What should be the Molarity of Primers for a qPCR after ChIP, the final concentration in a 25ul reaction. Some protocols suggest using 10uM stocks of each primer (0.5ul each), while other protocols suggest using 5uM each primer (o.5ul each) in a 25ul reaction. Which one is the best?
Thank you
Ikram
Hi Ikram,
In my opinion, the best concentration should be one that you have tested in your hand and works. For my ChiP assay, I usually use 1uM final concentration for each primer and this can be considered high but it has been working well for me. I would suggest testing a number of concentrations and based on the raw Ct for each primer concentration, you can choose which particular concentration would work best for your experiments.
augusta
I use 250nM which is pretty standard. Many protocols call for around 200nM. Although Augusta has a good point, I have never titrated my primers. This would cause a lot of extra reactions especially if you are doing a gene expression study with many different genes.
My stock primers are 100uM. I make a working stock with 20ul of forward and reverse with 760ul water. This gives me a working stock of 2.5uM for each primer. I consider this working stock 10X when making my master mix.
Dear Augusta and Dear Gary
Thank you for your suggestions. I have been using a working concentration of 10uM (each primer), of which I use 0.5ul each in a 25ul reaction, which means a final concentration of 0.2uM each. My raw Ct values are around 30 which is kind of ok for ChIP qPCR I think. I was a bit confused to see some protocols suggesting to use 5uM which would give a final concentration of 0.1uM.
By the way, since I have a huge set pf primers and different controls, titration would not be feasible.
Anyway, Thank you for your help. I appreciate this.
Ikram
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