After doing my primer efficiency, my lowest dilution cDNA ct value is 20.16 and NTC ct value is 31.76. The melting curve is the same for target product and NTC and it is giving the same product size in the gel. In spite of this, my primer efficiency is very good.
Is this situation OK? Please give me some suggestions. If it is not OK, what are the reasons behind it and what is the acceptable condition for NTC. Thanks in advance.
In the settings of qPCR, NTC does not have any DNA template in the reaction and its function is mainly to ensure that all reagents that you used (e.g. H20, SYBR Green or other qPCR reagent, and primers) are free of contamination and to detect the presence of primer dimer. It should not have any signal/Ct value as there is no template to be amplified.
As you are having NTC with an amplified product which has a similar size and even a similar melting curve as your target gene, I would say that it is possible that your reagents may have been contaminated with the template that you used to generate the standard curve.
Syed Monzur Morshed, it is unlikely since your amplified product for NTC has a similar size as your target gene. Primer dimer usually has a small product size at ~50bp. And it should not have a similar melting curve as your target gene. In my experience, primer dimer shows a melt curve before that of the target gene (it melts at a lower temperature due to its smaller size).
We also get Ct value for NTC but the melting curve is different as well as the band in normal PCR. Most of the times there is no band in case of NTC,unless the primers form dimers.So,I will suggest you to dilute the primers until it shows no bands in normal PCR, then use it in real time PCR.
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