Outer (edge) wells show systematically different absorbance values compared to inner wells, with CV >15%. Edge wells may show higher or lower signal depending on evaporation or temperature variation. Visual inspection from the side reveals lower buffer levels in edge wells.
Common Causes
1Edge wells exposed to greater temperature variations during incubation
2Outer wells experience higher evaporation rates due to humidity differences
3Plates used directly from refrigerator; inner wells take longer to equilibrate to room temperature
4Wells not sealed properly during incubation, allowing differential evaporation
5Edge wells dry out faster due to increased surface area exposure to air
Solutions
1Cover all wells with sealing film or tape during all incubation steps to prevent evaporation
2Allow plate and all reagents to equilibrate to room temperature for 30 minutes before use
3Do not use plates straight from the refrigerator; inner wells need more time to reach room temperature
4Fill outer wells with buffer only (not sample) and use only inner wells for assay if edge effects persist
5Incubate plates in a humidified chamber or sealed container to minimize evaporation