I am setting up qPCR with human DNA and actin primers, it is working with 10 and 1ng dilution but when I am considering 0.1, 0.01 and 0.001 I a getting same ct values for these 3 dilutions, I am sure it is not dilution problem because I repeated 4 times with proper care. I am also sure that DNA and primers are fine.
Some one please help me.
Is the melt curve good (single peak)? Also, what is the CT value you're getting? And are the primers validated or are they newly designed?
I've had non-linearity issues with some primers for my RT-qPCR work. Usually re-designing the primers helped resolve the issue. In some cases the primers with linearity problems also showed signs of non-specific amplification (more than one peak in the melt curve.
Most likely there is contaminating template in one of your PCR reagents (e.g. oligos or dH20). Alternatively, your primers are misamplifying as Brian suggests. If you've used these oligos successfully in primer experiments, I'd start with fresh reagents/stock solutions. If not, then check your melt curves to check on the specificity of your qPCR results.
Agree with both previous comments. I would run a gel and check abundance and purity of products. Using another set of primers targeting a different gene in the same target species would be another way to check.
Hi,
melt curve looks fine (Single peak), I used fresh stock, no difference. Even I ran the sample and primers on the gel, looks fine. If one or two bases are gone bad in the primers I cannot detect on the gel. Anyway ordered new primers.
Thank you all..
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