Your amplicon size is too short. it should be 120-150/200,

if normal PCR worked perfectly than please check you sybrgreen master mix. Does other primer give appropriate ct value with same master mix?  

Hi Shukkoor

Have you checked your primers in silico? I took a quick look at IDT's Oligo Analyzer and I saw some primer dimers that may be of some concern. Considering you have an amplification on your NTC and the in silico prediction I believe your primers are forming dimers. Another thing that I noticed is that the predicted Tm for these primers are around 50oC so the higher temperature may compromise your qPCR amplification. As for the band you see on your gel, the size of small fragments is difficult to caracterize using agarose gels.

In conclusion, I believe you should redesign your primers.

Tajas and Andy, 

We did choose a primer set from a chemistry paper published in JACS, they amplified 100 bases, we have 85 base ssDNA. Tm of the primer in qPCR salt concentration is approx 60. So that may be also not an issue. We even tried 50 oC reaction but the result was same.

Shukkoor

After your reply I checked again on Thermo Fisher and IDT websites, both of them said the Tm for the primers (with 50mM salt concentration and 300nM oligo concentration) is around 50oC.

One thing that I noticed is that your reverse primer is wrong (it's 3' / 5' instead of 5' / 3'). Did you order it this way or did you order it correctly? Also, did you run a melting curve on your amplification?

https://www.thermofisher.com/br/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html

https://www.idtdna.com/calc/analyzer

I also get the Tm 50 this way, but when I opt for the qPCR in IDT instead of the default it gave me 58.

Also the second I assume the RV primer should be reverse compliment ?

Melting curve was done and so Tm around 75

You are correct the reverse primer must be ordered as reverse complement it was my mistake.

How was the melting curve? Did it have one single peak or two?

Do you a picture of the amplification curve and also melting curve?

We did get single peak 

Shukkoor

That is very (VERY) strange. I've never seen straight lines like that. It's like the fluorescence is high from the start without any amplification.

You are conducting qPCR from sinthetic ssDNA, right? I believe the reaction efficiency is higher than gene expression qPCR. Have you tried titrating your ssDNA and primers?

In the second image you do have some amplification but with a high Ct indicating you should use less template. Your melting curve are looking very good, that don't look like dimers. Which device are you using? Are you setting the baseline manualy or automaticly? What does it happen when you change the base line?