I have replicated my experiments a number of times but results are coming like this all the time. I am not getting any Threshold cycle. It would be of great help if anyone could give me some suggestions regarding this.
Yikes! That is a really messy graph. Are you certain that the machine is calibrated properly? You should see zero (or very close to zero) fluorescence for at least the first 10 or so cycles.
And you should never see the signal drop once you start to get amplification.
Might send this to tech support for the thermocycler company.
Check for the possible contamination of nuclease-free water or any other experiment ingredients.
Thank you so much Govindkumar Balagannavar and Katie A S Burnette for your kind suggestions. After a bad patch of failing, I have finally obtained the perfect amplification curve today.
That amplification curve does not look perfect. You have different slopes (e.g. different amplification efficiencies) for at least 2 different products.
Check that your primer efficiencies are 95-105%.
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