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Hello Stack Exchange Biology world,

I have struggled with my qPRC results for over 5 months. I have a bunch of DNA samples, and I want to find the quantity of Pseudomonas syringae contained. My issue was the extremely high efficiency (sometimes even 1200%) after the run. So, I decided to make everything from scratch. I used a new set of primers and a new qPCR kit (kapa sybr fast qpcr master mix (2x)), and I switched the sterilized double-distilled water from the water filters in the lab to an ampoule one (to eliminate any contamination). I incubated the bacteria again. I measured the OD=0.998A, before extracting this DNA (via NucleoSpin Soil, Mini kit for DNA from soil) with the following quality as 193μg/mL, 260/230 = 2,41 & 260/280=1,89, which seems high quality.

For the standard curve, I did dilutions of 1:10, 1:100, 1:1000, and 1:10000 using the proportion of 5μl of DNA with 45μl SDW from the ampoule.

So now I have everything new and a "high quality of DNA" to work with. Yesterday, I attempted to perform a qPCR (SYBR green), and the efficiency was -100%. What should be wrong at this time? Is it something that I can change in my 20μl reaction?

SD Water from ampoule 7μl
QPCR Master Mix 10μl
Primer F 0,3 μl
Primer R 0,3 μl 
ROX high 0,4 μl
DNA 2μl

The machine I performed the reactions on is the Thermo QuantStudio 5 Real-Time PCR. Below, I am providing the experimental details as well as the qPCR results.

Thanks in advance for any suggestions to help me fix this issue. enter image description here enter image description here

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  • $\begingroup$ Normally, if your qPCR efficiency was negative 100% I would say your dilutions were made properly just entered into the software in reverse order. But your amplification curves are pretty funky. How are you setting the threshold? And what is the baseline determination? What about the melt curves? The protocol is strange too, in that it's taking multiple fluorescence readings per cycle. I wonder if that's screwing with the baseline readings. Typically just one measurement is taken after annealing/extension. Have you tried a 2-step protocol (deleting the 79C step)? $\endgroup$
    – MikeyC
    Mar 6 at 16:34
  • $\begingroup$ @MikeyC Thank you for your response. As I'm learning qPCR, I combined the KAPA SYBR® FAST Master Mix with a P. syringae protocol I found. journals.asm.org/doi/10.1128/aem. The main issue was that the protocol used a different qPCR mix and annealing at 54°C compared to KAPA SYBR®(annealing at 60°C). I adjusted accordingly, but perhaps incorrectly. About the threshold and baseline determination, I used default settings. For my next run, I will only keep step 2 for fluorescence reading as recommended. Any further suggestions for refining my experimental method will be highly appreciated. $\endgroup$
    – KGee
    Mar 6 at 19:47
  • $\begingroup$ Run some of the qPCR results on a gel or have a look at the melt curves - you might well have more than one product being generated. Your std curve should have a little over 3 cycles between each point (copy number doubles each cycle so 3 cycles is 8x more copies to reach Ct) and the replicates should be quite tight. How long is the PCR product? It shouldn't need extension if it is <100 bp. $\endgroup$
    – bob1
    Mar 6 at 22:12
  • $\begingroup$ @bob1 I did, and you are absolutely right. Indeed, I have two zones on the curve amplicon. How do you know that? Wow, I am just surprised. Does it mean that my primers were not extended correctly? The primer sizes are 194 and 162 bp. $\endgroup$
    – KGee
    Mar 7 at 19:53
  • $\begingroup$ @KGee It was a suspicion (MikeyC noted it too), experience is a great tool... Your primers are 194 bp and 162 bp each? Or are those the amplicon sizes? Primers should only need to be about 18-25 bases for a highly specific reaction. The manual for the Kapa pol says extension of products less than 400 bp should need 1 second. $\endgroup$
    – bob1
    Mar 7 at 20:00

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