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The efficiency of my standard curve is not within 90 – 110%. What troubleshooting suggestions do you have?

Inspect the curve for outliers. If any of the data points appear to be far from the fitted curve, delete that data point and see if the efficiency improves. A minimum of three standards is required for a reliable standard curve, so up to two standards can be deleted entirely. However, each standard needs at least two measurements, and only samples that fall within the remaining standards can be quantified.

Inspect the No Template Control (NTC) to ensure it has a greater Cq than DNA Standard 5. If the NTC falls within the standards’ range, there may be contamination present in the standards or reaction mix. This may come from other samples on the plate, or traces of other libraries remaining on laboratory surfaces or equipment. Clean all instruments and surfaces thoroughly and repeat the experiment with fresh aliquots of reagents.

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