I am attempting to perform a qPCR standard curve using viral DNA (Adenovirus and MCMV) and cDNA (Influenza A) to be used to determine relative abundance of these viruses in primary samples. Viral DNA and RNA was extracted from viral stock using the QIAmp MinElute Virus Spink kit and vRNA was converted to cDNA using the ImPromII Reverse Transcription System. 6 point 10x dilutions were performed using this viral DNA and cDNA (undiluted-1:100,000) and a negative water control was included. For the qPCR, I am using TaqMan Universal qPCR Master Mix along with virus specific primers (forward and reverse) and probes whose sequences were provided from literature review or provided by neighboring labs but have not previously been used by our lab with this particular master mix. I believe that all of the input DNA/cDNA concentrations are fine (ranging from 17-180 ng/rxn note: master mix prefers concentrations
Hi!
Could you please include the photo of your melting curves from this experiment? You're using BioRad machine, I believe, the melting curves can be found in one of the tabs.
(It is possible that your target is not amplified at all, judging from amplification curves; melting curve can give me more information about that).
Also, if you still have the plates that you ran, try running the finished reaction on agarose gel and see if there's the expected band (the gel should have very high agarose concentration, like 3.0 for that, since your fragments are so small).
Yulia Panina if you work with the Taqman you don't have the melting curve.
@Ferralita do you try to made a qPCR with Syber, only to compare the efficency of the primer? Only to test if the primer work properly. Or is possible to check this in a PCR.
Hello Jack Greenhouse , I have mapped the primers and they appear to completely match with the Influenza A sequence. I am using H1N1. I have not used these primers for anything except for these qPCR experiments. A neighboring lab has used them successfully in the past for qPCR, however they used a different master mix. Thank you for your suggestion.
Hi Giorgia Pertile , I have not tried the qPCR with Syber. I have used the primers for PCR to amplify the Adenovirus and MCMV targets prior to qPCR and there was amplification of these specific targets. Please see the attached photo.
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