Hi,
I often hear (and say) that tubes should be put at the center of the PCR machine for a better reaction performance. I was wondering if the same applies for qPCR and therefore if there is an effect of the well position on the ct (I'm using the BioRad CFx96 real time system).
Thanks,
Fabien
If you have an older PCR machine you may see more 'edge effect' (difference in gradient and temperature across the plate) which, dependent on the primers/target/amplicon, may or may not are more not give rise to slight changes in efficieny of the PCR reaction. This may affect ct but then ensure you run controls at various positions across the plate to help you discern whether or not chnages in ct are real or an artefact.
Normally there is no effect on ct for well position. But be sure that your machine is working perfectly.
I personally think there should not be any significant difference between wells but the 'edge effect' may happen if the seal or cap-strip cannot seal the plate properly. Severe evaporation may happen to the wells on the outer area of the plate. Some suggests the combination of heal seal and two-component plate may greatly reduce the effect.
Thank you all for the answers, I guess I'm getting a bit paranoid with my qPCR experiments.
I've seen it in a very old machine, but modern ones with LED light sources are much more reliable. You are much more likely to see an edge effect from a plate that isn't sealed properly and has evaporation from some wells.
It's easy to paranoid about qPCR - totally normal!
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