I just started using qPCR (learning the logic and the basics, actually). I am trying to create a standard curve for ecoli and Pseudomonas syringae. I started by making some DNA dilutions of 10%, 1%, 0,1%, and 0.01%, and I amplified them along with the non-diluted DNA (let's call it zero dilution) and three replication of each DNA.

In my output (just an example), I got a curve in which some dilutions are a kind of mixing, let's say: zero dilution comes first, followed by 10%, then 0.1% (instead of 1%), then the 1%, and lastly, the 0.01%.

Actions made: I checked the CT values (I got cts for all my amplifications)

I loaded the amplification products on agarose gel, and I got zones in all of them.

Question: Why do I have the least diluted DNA solution before the more diluted one?

Thank you in advance.

PS I am not a microbiologist, so my knowledge is limited :P. Of course, any suggested paper that explains this issue is highly appreciated.

  • $\begingroup$ The obvious thing is that you have made a mistake of some sort. Exactly what that mistake is is difficult to say - possibly technical (e.g. reaction efficiency) or possibly something wrong in your set-up. Get someone experienced to show you how to do this. $\endgroup$
    – bob1
    Jun 23, 2023 at 10:47

1 Answer 1


Probably easier to help diagnose a problem if you upload a screen shot of the amplification curves and/or melt curves. One issue that a lot of people run into when starting out is that the dilution process is going to be very sensitive to contamination. So, you need to be mindful of what DNA templates have been handled near your workstation in the past and always change tips between each dilution step. Even the smallest droplet of your neat DNA (your "zero dilution") could contain enough template to dramatically change the Ct values in the 3rd or 4th dilution of a 10-fold series. This also can apply when loading your template dilutions into your plates.

One simple way to check is to plot your Ct values against the log of your relative template concentrations and run a simple linear regression. The points should all fall on a straight line with slope around -3.32 and r-squared of 0.98 or better (obviously they won't at first in your example case). If you swap the values for your out-of-order samples (1% and 0.1% in your example) and the regression line straightens out, you probably just mixed up those dilutions when loading your reactions. If you swap them and you still don't have a straight line, you probably made a mistake in the dilution steps. Either way, do it again to confirm.

This all assumes you're have a well validated primer/probe set, adequate starting template amounts, appropriate master mix, and appropriate controls. If your starting template is on a plasmid, it should be linearized or digested to avoid supercoiling, which can make for weird qPCR signals. If you're not using filtered pipette tip, use them. Never handle amplified templates in the same area you set up your plates, and never use the same pipettes for both.

This link has a collection of papers on qPCR that might be helpful for getting started. https://bitesizebio.com/62143/qpcr-papers/


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