In the qPCR I'm performing with Biorad cfx 96 with Biorad universal master mix, I am trying to amplify the 62bp amplicon region with the standardized temperature settings. The probe is FAM labelled with TAMRA quencher. Final concentrations of FP, RP and probe are 100nM, 100nM and 200nM respectively. The primary experiments shown NC amplification after 30 cq. But in the later exps., ran along with the stds, the NC ct value is lesser than 18 and far ahead of the highest standard 10 power 7 copies (cq 21) we used. Further gel analysis was done and the amplicon was confirmed as expected product (Gel pic attached).
1. Is this contamination? How is it possible to get an contamination with the highest copy number the lab never used?
2. If the reagents are contaminated why all the stds and NCs did not showed same cq?
1a) Almost certainly yes, it is contamination.
1b) The highest standard that you prepared may have 10^7 copies, but the number of copies in any positive PCR reaction well could be 1000 to 1000000 times higher. So it would take only a very tiny drop of such PCR product to give you this result. This would appear to be classic "carryover contamination".
2) This must have to do with exactly how the reactions were set up. Is there any component that is added only to the NC reaction? If so, it is likely that this is the component which has the carryover contamination.
An amplicon of 62 bp is a problem for more reasons. It cannot hardly be disciminated from so called primer dimers. Moreover it is difficult to sequence without adding langer additonal sequences.
But you must try forward-reverse sequencing to identify the product as carry-over contamination. Otherwise you cannot say its' the amplicon in your NTC.
If it is indeed an amplicon you have a big problem.
Elizabeth makes a very good point. And it is often true that the strongest yield of primer dimers will occur in the no-template control. So there is an issue of whether the band that you are seeing is actually a true PCR product.
If you cannot sequence; try to find a probe within the amplicon
If your NTC does not react; it's likely a primer dimer. Did you perform in silico analysis of your primer pair? What was die delta G of the 3' ends? Whats the internal compmentary delta G? Etcetera
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