Dear Mariano, what I could suggest is to 1. Use different concentrations of your samples. May be to start with more crude (concentrated) sample followed by 10 fold dilutions. sometime the problem is due to too low or high copy number of your target gene. 2. you need to check the Tm of your final product and calculate whether it is the desired Tm for your final product. Hope it works  

Try to use equal amount of template DNA every time.. and the DNA amount should not exceed to 40ng in your reaction.. 

 Hi Harsh and Ramgopal. Thanks for your answer. I comment to you,

my standard curve are five points 1/10 dilutions starting with 50 ng template DNA, I always use the same amount of template.

The first point is 8 Ct, some people told me that is too early, but between 10-30 Ct is where I have more problems.

I check the final product Tm and the melting curve is fine (one peak), the problem is the separation between the dilution.

I attached a picture of a good run, but you can see the efect I discribed to you. notice the difference between the first 3 and last 2 dilutions.

Thanks again

Dear Mariano, Thanks for the graph. 

Like others, I too agree that Ct8 is too early; this may be explained by the high expression/copy no of your target gene. You can further additionally (1) dilute your sample, leaving your first dilution

Second, as per the picture attached, there seems to be no such problem. perfect replicates. What about your standard curve. Are you able to plot a reasonable std curve with good efficiency

As you mentioned earlier, you are getting variable results; this may be sometime due to improper activity of few wells of your real time machine. We faced this. sometime all wells are not read perfectly. So you may switch over to some other segment of your block or may even try another equipment 

Hope it helps

Hi Harsh, thanks for the answer.

To check the linearity I test 8 point 1/10 dilutions curve and always the best 5 point were the first ones because the effect I described earlier, the dilutions begin to separate more and more. 

I attached now a curve that it seems to be fine but the efficiency is 74 %, R2 0.992. Sorry the bad quality, I have to take a picture with my phone.

I check the final product Tm an is 79.3, do you think I have to increase the T° in the final step? I used 72 °C because it said in the certificate of the master. Maybe the  template is not completely denatured.

Thanks again

Dear Mariano 

As per your standard curve and amplification curve, to the best of my knowledge, I could not figure out any problem. Moreover, your R2 value is also perfect. Can you re-check the fold dilutions you are providing to your cycler software for standard curve calculation i.e. 2 fold, 10 fold etc. This may be the cause.

Also, I would suggest you to use 2 fold dilutions, starting from 100,50,25,12.5,6.25,3.1251.56 .....; they work equally perfect for us.  

What do you mean by the final step: if you are talking about melting, thn it is continuou and goes till 99oC. 

As per your last amplification plot, you should get two Tm, One for your product (Ct below 20) and another for primer dimer (amplification visible after Ct 30)

Hi Harsh,

I know it´s seems OK but it has a 74% of efficiency, maybe in the picture it doesn´t seen but the dilution are separate for 4 Ct.

I´ll try the 2 fold dilution, I was using 10 fold dilutions.

The final step I mean in the cycling part, not in the melting (check my first question). I´m thinking if maybe it a problem that the last step is at 72°C and the final product Tm is 79.3°C.

The Tm that I pass you is the final product, I attached the melting graph

thanks