Hi
I'm trying to design a multiplex for COVID-19 detection. Whereas I'm seeing an unusual challenge of a pseudo raise in all 3 viral genes and in the internal control genes between cycle 07 & 09 followed by actual amplification plot as predicted between 25 & 36 cycles. I repeated many times and adjust the annealing temperature as well. Slightly adjust the primer/probe concentrations. Primer concentration ranges 150-250 nm and probe in 100-150 nm. Why I'm facing this issue and any recommendations to solve this? Attaching the amplification plot files as snap shot.
Also I adjusted the background subtraction curve fit options. This error is not showing if we are working on other assays.
hi,
some questions:
- Did you check the raw data for the same drift? You can change it under baseline setting.
- Which Mastemrix are you using? Are you mixing differently?
- Which reaction volume are you using?
Best Sven
Srinivasakumar Kp Hi, if you want to just check the viral RNA expression in sample then use N2 primer-Probe set.
In both which method you have used TaqMan method or SYBR?
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