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What should I do if my standards replicates show significant variation?
A variation of ± 0.2 cycles between replicates is normal. Check the melt curves to ensure there is a single product peak. (There may be a lower molecular weight shoulder present, this is normal). If there are multiple peaks, this suggests the standards have become contaminated or degraded. If that standard can be removed from the analysis, do so. Otherwise, order new standards and repeat the experiment.
If the melt curves show a single peak that overlaps between replicates, the variation is probably caused by inaccurate pipetting. Repeat the experiment, taking care to pipette the standards accurately.