Hi, thanks for your answer!

In the experiment shown, the dilutions are from 10^6 to 10^2. Previously I have tested 10^10, 10^9, and 10^7 and the curve starts skewing (a short "plateau").

Maybe I can add 10^7 also? that would be 7 points for the standard curve.

The thing is that the R2 is superhigh, often 0.99...

How is it possible that using a combination of primers with the lowest Ct (500 nM of each), the slope is still -4.2??

Could be that I'm separating annealing (58C) and extension (72C) steps instead of doing one single step at 60C?

The problem has probably very little to do with you using separate annealing and extension temperatures.

Looking at your graph, it appears that the highest concentrations are the problem. With a dilution of factor 10 you would expect a difference of about 3.3 in threshold cycles. Try fitting separate curves to the highest three and two the lowest three concentrations and see if there is a difference.

If there are other issues I would assume your problem is either in the primer design or with inhibitors from your sample. What is the annealing temperature of your primer? From my experience with 58C is a little high, maybe try decreasing it by one or two degrees. You could also try adding a little DMSO (about 0.5v%) to your reaction, sometimes this helps.

Hi Christian,

thanks for your answer!

The annealing temperature is 58C but I also used 60 C with the same results.

Do you think that the fact of that the DNA standard is a circular plasmid is important?

Do you think I need to change primers?

thanks!

Hi Dr Huerta,

From your amplification plot, I assume that this may be due to supercoiled plasmid, which you are using as a standard template. Suercoiled templates are well known to interfere with efficient PCR, particularly in case of lower copy number reactions, which seems to be true in this case too. So try using linearised plasmid (single RE cut distant from your primer binding site) as standard template. I believe that your qPCR efficiency will improve as well as your replicates will also be more relevant. You can go through the following threads & published material, including one of our works on template topology and PCR efficiency.

best wishes,

SD

https://www.researchgate.net/post/Untreated_extract_plasmids_as_templates_in_qPCR?_iepl%5BgeneralViewId%5D=nPIlepAX2O4jCGPmwc3HxD6YjQOjTYEv8kpN&_iepl%5Bcontexts%5D%5B0%5D=searchReact&_iepl%5BviewId%5D=NPQNqyLAhPF11NkQSV3URIayRrwl70NF7WBL&_iepl%5BsearchType%5D=question&_iepl%5Bdata%5D%5BcountMoreThan20%5D=1&_iepl%5Bdata%5D%5BinteractedWithPosition12%5D=1&_iepl%5Bposition%5D=12&_iepl%5BrgKey%5D=PT%3A58fa184d96b7e4da477641e7&_iepl%5BtargetEntityId%5D=Q%3A58fa184d96b7e4da477641e7&_iepl%5BinteractionType%5D=questionView

https://www.researchgate.net/post/Why_is_it_better_to_linearise_a_control_plasmid_before_PCR

https://www.researchgate.net/post/Plasmid_DNA_vs_cDNA_for_qPCR_standard_curve2

https://www.researchgate.net/post/taqman_probe_real_time_PCRProblem_regarding_constructing_plasmid_dna_standard_curve

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832698/?tool=pmcentrez

https://academic.oup.com/nar/article/35/4/1377/1749276

Article Enhancement of PCR Detection Limit by Single-Tube Restrictio...

Thank you so much, Dr. Datta!

I purified a PCR product from the template and diluted it serially.

Now the efficiency of one of the primer pairs (blue curve of the attached jpg) is 93%, with R2=1, so apparently it worked! ;-)

For the other pair of primers (green curve; efficiency 75%), I have still to do some optimization with gradient PCR and combinations of primer concentrations.

Again thank you so much for your advice!

I hope with some optimization your other primer can also give you better efficiency. It seems that the amplicon generated by the primer with green curves are longer than the amplicon with blue curves. Please try to keep the primer specifications within the manufacturers recommended range. Things will be fine. :-)

You have done right. Actually I forgot to mention that apart from linearized plasmid, properly purified, quantified and diluted amplicon can serve as very good standards. The only issue with linear standards is their stability. However you can make fresh linear standards and use them within a week or so without any significant variation.

Best wishes

SD