I have a target of 176 bp which got a Ct value between 24-25 (three replicates), i also ran gel from PCR amplification and from amplicon of qPCR after melting curve
melting curve has a sinlge peak but both gel shows no band at 176 bp. There is hazy band at below 75 bp....i consider that primer dimer....this primer dimer also exists in gel from amplicon of qPCR after melting curve (which i should not expect?)
Any explnation!
Ct value is the interpolated cycle where the sample crosses an arbitrarily defined fluorescent threshold value. It is (relatively) representative of the concentration of the starting material, but onto itself not a measure for DNA quantity. If you set the fluorescence threshold low, the samples will cross that threshold at a low Ct.
Have a look at your amplification curves to see if it plateaus at your final cycle (35, 40, 45). A plateau would indicate that no further amplification is occurring. If plateaus do occur in the wells that you are pipetting onto gel, you could combine several wells of the same amplicon to get a stronger signal on gel. You can also increase the amount of DNA dye in your gel. We use 1/20.000 midori green to visualise DNA, but for dificult to see amplicons you could choose to increase this to 1/10.000. Good luck
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