I'm performing a real-timePCR experiment (Applied Biosystem step one plus) using SyberG as fluorescent dye and ROX as passive dye. At the end of the run, only in some wells, the software gives me an error: BAD ROX. I have noticed that in such wells, during the cycles the signal of the ROX decreases. I don't know how to explain...
Your sample may be evaporating (?). Are you using a low volume of the reaction? I never have had that issue. But, would want to know, what it means.
Its an interesting situation, but you need to sure your step one plus all calibrations are up to date, let your machine checked thoroughly by Applied Bio systems tech. people, usually such problem may occur by any misalignment within the unit..so some wells may give such bizarre readings in such instance..So just re-calibrate your machine again and check if there any warning to clean some wells of thermal block so simply clean them by lint free lab papers in 70 %Isopropyl alcohol.
I'm using a 25ul reaction volume. The machine has just been bought. Specialists say that this phenomenon happens when the target gene is highly expressed, however I've never had a similiar problem. I used an annealing temperature of 53°C (the optimum temperature for my pair of primers), hovewer i noticed that using a 60°C annealing temperature the problem disappears.
check the melting temperature, maybe some problems with specificity
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