I tried some chemical modifications on a primer sequence and tested them against the unmodified material. Actually, I just was about to check the ct value to see if they still were usable. However, at higher cycle numbers I found differential behavior of these primers, these results are reproducible (2 independent experiments with duplicates). Please refer to the attached screenshot. (the blue and the green curves are the no template controls).
After the first peak, I attribute the decay in fluorescence to the limiting FRET probe which might have been used up, but have no clues why the signal later behaves differently, as the amount of PCR product should be approximately the same, according to the similar ct values (around 13 +- 0.5).
Any conclusion I might draw from the different behavior?
Template and 2nd primer were included in the mastermix, concentrations of the first primer were adjusted according to UV measurements after recovering them from the chemical treatment.
PCR conditions: 0.5uM primer each, 0.4uM FRET probe each (final conc). 0.1nmol template (approx 100b singlestrandred oligo), total volume 20ul.
Any ideas, suggestions?
Chemical modification of primers are usually coupled with loss of specificity as well by increase in cycle number. Probably this double effect deforms your curves. You can try to dilute your primers or any modifications increasing the specificity...
Dear Szabolcs,
that's exactly what is puzzling me: If I had loss of specificity or even degradation, I expected to get differences in ct values, not later, when I assume that some of the limiting factors of the detection reaction already should have been used up.
I think the peak around 0:30:00 is true positive. Blue and green lines are negative. So, the peaks after blue and green line may be negative. If you use hybridization probe, I suggest using TaqMan probe. TaqMan probes probably reduce non-specific reactions... Good luck.
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