Hi,

I have a short amplicon that is 53 bp long. I want to quantify how many DNA copies in my NGS sample by amplifying the short amplicon with TaqMan qPCR method.

My probe is 20 nt long, forward primer is 17 nt long, and reverse primer is 20 nt long (Total 53 nt). I always see amplification in NTC (No Template Control). I confirmed that there was no contamination while preparing the qPCR experiment.

Even when I performed a qPCR with SYBR Green method, I was able to see a high amplification in NTC. I remember the Ct value was between 22 - 26. In my opinion, there is primer dimer formation in NTC sample.

I designed 4 new pairs of primers to overcome this problem, but I could not. Due to the length of the short amplicon, I could not design many.

Here are the details of my qPCR below.

[My qPCR details]

- Machine: StepOnePlus v2.3

- qPCR kit : TaqMan™ Fast Advanced Master Mix (no UNG) - I have RNAs in my NGS samples.

- Mode: Fast mode (~40 min) and qPCR standard curve

- Concentration of primers: 900 nM

- Concentration of probe: 250 nM

- PCR condition: Hold at 95 C for 2 min and then 40 cycles of [95 C for 1 sec / 60 C for 15 sec / 72 C for 1 min]

Can anyone provide me any suggestions to overcome this amplification issue in NTC sample?

Thank you.

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