Hi,

• It depends on several factors. First, try to determine how much reaction you want to run. The primary concentration of your qPCR reaction can range from 15 to 30 microliters. The qPCR machine is also can affect it. For example, MIC real-time PCR apparatus enables you to run 10 microliters of your reaction, consequently, you can save a considerable amount of input chemicals per run. Corbett rotor gene 6000 real-time PCR is also working well with a lower concentration of your qPCR reaction.
• A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM. For dye-based qPCR and Real-time PCR the optimum dose of each R/F primers is mentioned in their protocol and you can select the best concentration for your goal.
• Before running all samples, try to set up your reaction with some primary runs and using different concentrations of R/F primers.
• Primer design is also a quintessential step in your project. If you are not an expert in molecular biology, somebody other may support you for this case. Nevertheless, if you want to do it selfishly, I highly recommend you to use PerlPrimer, an excellent user-friendly and versatile PCR primer design software. You can get it through the following link:

http://perlprimer.sourceforge.net/

Our lab generally uses 200nM concentration of primers but it can range from 100 to 500nM.

Another alternative would be to use IDT Prime Time primers. They come up with 20X Prime Time primers which is to be diluted to 1X for qPCR in 20ul reaction.

I believe a good starting point would be primer concentration in reaction 0.5 uM= 500nM. Which means 1ul of each primer (previously diluted to working concentration on 10uM) + 5ul sample + 3 ul water and 10ul of 2x conc mastermix. To asses ideal concentrations you can use checkerboard (e.g. https://www.sigmaaldrich.com/technical-documents/protocols/biology/primer-concentration-optimization.html) . Make sure to have a good positive control fro optimisations. Don´t be surprised if the result of optimisation is uneven concentration of primers - they may differ in annealing temp., length. hope it helps.

Thank you very much with the help everyone!

Hello everyone!

I have designed a few primers and then a tried optimized the conditions using reference RNA, diferente primer concentrations e annealing temperatures and then calculating the primer efficencies using a standard curve. Finally, i think i have my PCR conditions optimized and the efficiencies for each pair primer are > 90% and < 110% as recommended.

Now i want quantify these genes expression by qPCR in my samples. Do i need to always perform again a standard curve in each plate?

I'm using SYBR Green and differences in gene expression will be calculated using the Livak method.

Hi,

Generally It is 200-500nM. But primers can also be titrated individually to get well optimized assay.

Final concentration 0.25 μM

Our lab uses 0.5 µM

It varies to a reaction to reaction and machine, I was used 1uL/rxn of each primer for 10uL reaction (Sybr 5uL, Primers 2uL, Template 2uL, etc).