qPCR: high replicate ct cv

1. Ranked causes

   1. Master mix incompletely mixed before dispenseCV increases toward later-plated wells; vortexing before dispense helps.
   2. Baseline drift on a subset of wells (analysis)Raw curves look similar; Ct disagreement shrinks with shared baseline settings.
   3. Inconsistent pipetting of master mix or templateCV worse on manually plated wells than on a calibrated dispenser.
   4. Evaporation / edge effects on the plate during the runOutlier wells sit on the plate perimeter; optical seal incomplete.

2. Recovery steps

   1. Re-plate with reverse pipetting; discard bubbled wellsVolume inconsistency is the top driver of technical Ct CV.
   2. Vortex master mix immediately before every dispense roundPrevents settling across a long plating session.
   3. Replace optical seal; avoid leaving edge wells empty without buffer moatReduces evaporation-driven perimeter outliers.

3. Controls first

   • Are replicate CVs high on standard-curve / positive-control wells as well?If yes: Pipetting, mix, evaporation, or analysis — not sample biology alone.

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