Hi I am currently doing a quantitative real time PCR on my samples. I am quantifying the viral load (WSSV) on different crustaceans using the product KAPA SYBR FAST qPCR. I just want to ask if anyone here experience using the same product for quantifying DNA? May I know if you are adding dUTPs on your samples. This is because I've tried adding dUTPs on my master mix and the melting curve went well. However when I tried to use the SYBR FAST mastermix only (w/o dUTPs) I'm getting two peaks on my Melting curve. I know that getting two peaks on melting curve maybe due to the primers, but how come when I added dUTPs on my mix the melting curve went well?
Generally, it's no problem to use dUTP instead of dTTP. The DNA-polymerases used in PCR will not distinguish these nucleotides. Using dUTP is rather common, as UNG is used to keep cross-contaminations of PCR products under control (you can google "ung mastermix" to read more).
The melting curve can be used to check for correct amplification only after validation. That is, during the validation pahse of your assay you must run gels as well - then you can correlate the observed Tm with the bands occuring on the gel.
Sir @Jochen I've tried the the master mix without adding dUTPs and these are the melting curve and quantitation results. As you can see, my NTC exhibited a peak as well as my 10^3 standard. I've tried to run a gel after this and got no band on the same samples (NTC and 10^3). This confirms that the peaks are primer dimers. My problem now is the quantification of DNA. As you can see, the NTC also exhibited a quantification even it is a primer dimer. What should I do for this? Thanks!
Thank you for your immediate reply Sir Jochen!
Another question, is that the standard Ct value (
That's just following from your melting curves. The 106 standard showed specific amplification, and its Ct is 24.1. This allows to conclude the inverse that if you observe a Ct of 24 (or 25, too, surely) that the amplification is specific. If your samples give you Ct values lower than that you are on the safe side to take their Ct values for quantification.
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