Setting up the plate: layout before pipetting

Standards in duplicate or triplicate

The standard curve converts absorbance into concentration, so its quality sets the quality of every result on the plate. Run each standard level at least in duplicate, in a full serial dilution series with no gaps, and put the series in the same columns on every plate so runs are comparable. Replicates that disagree flag a pipetting problem while there is still time to catch it. Triplicates tighten the curve further and are worth it when sample numbers are low or the assay is new and still being characterized.

Blanks and controls, placed consistently

Blanks tell you what the plate and reagents read with no analyte; controls with known values tell you whether the run worked at all. Give both fixed positions and keep those positions from plate to plate and week to week, so a drifting control is visible as drift and not mistaken for layout noise. Include a positive and negative control on every plate, not just on plates you expect to question later. When a run looks wrong, the controls are how you decide whether to trust, repeat, or troubleshoot it.

Sample layout and dilutions

Plan sample dilutions on the map, not at the bench. If samples need diluting into the range of the curve, lay the dilutions out so each sample and its dilution sit in a predictable pattern, and keep replicate samples in different plate regions rather than side by side, so a local problem such as a bubble or a wash defect cannot produce two matching wrong values. Record the dilution factor on the map itself. A result without its dilution written next to the well position is a result you cannot reconstruct later.

Edge wells and edge effects

Outer wells evaporate faster and often run at slightly different temperatures than the center, which is why edge wells can read high or low compared with identical samples inside the plate. If your assay is sensitive to it, keep standards and controls off the perimeter, fill unused edge wells with buffer or water to even out evaporation, and seal the plate during incubations. Whatever you decide, decide it the same way on every plate; an assay that sometimes uses edge wells and sometimes does not is quietly running two different assays.

Related guides

• the 384 well plate guide: formats, a 384 vs 96 comparison, and templates
• the 96 well plate guide: types, volumes, and a free plate map template
• best deep well plates by job, plus sealing and KingFisher fit guides
• the cell culture plate guide: treated vs untreated, formats, and surfaces

Common questions