I am working on quantstudio 5 system with diagnostic test kit - AZF Microdeletions REALTIME PCR genotyping kit (master mix is aliquoted to every separated pcr tube, I only added taq polymerase, mineral oil and DNA for pcr preparation step)
After doing several tests, i found some abnormal amplification curves (heaved curve with a small rise at very end pcr cycles) (attachment picture).
Does anyone know what 's happened?
Besides, the threshold on the picture is set automatically by QS5 system, can I trust this threshold to determine whether my pcr target presents or not?
I am really appreciated for your help.
Thanh Tran It seems like there is no amplification and the primers are not working. You can compare this plot with your "no template control" sample, if that is similar to this plot shown here then it validates primers are not good for quantification or the qPCR condition is not optimized. You can also do a melting curve analysis which may show an increase in fluorescence might be due to primer-dimer.
@Sayanta Bera picture below is my negative control and sample. What do you think about this?
What happened is that you have 0 amplification in your samples. Look at the scale on the Y axis of your plot. The data are being stretched to fit the size of the screen. That is exaggerating the fluctuations below the baseline - it's just an artifact of the way the data are presented.
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