I am having a problem with the reproducibility of the endogenous controls of my samples in qPCR. I have tested several concentrations of cDNA with the objective of discharging that this was the problem and I have found that for all concentrations the amplification cycle is similar, which leads me to think that I have the reaction saturated.
At this point, should I decrease the amount of syber or primers or both?
Hmm, usually SYBR is used at a set amount, and should not be influencing the Ct. Primers are usually in excess so as not to be a limiting factor.
My first thought is that you have very high expression of your positive control gene(s). Try a 10-fold dilution series of your cDNA (original, diluted 1:10, diluted 1:100, diluted 1:1000, diluted 1:10,000), this large of a range should give you differences in the Ct value.
If that doesn't work, could you post pictures of your results?
Do you run a no template control sample in your pcr and is it failing to amplify as expected?
Yes, I included both the cDNA amplification control and water and for both there was no amplification, so I rule out the contamination problem.
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