Expected Results - NEBNext Library Quant Kit (E7630)

5.1 Typical results from a NEBNext Quant Kit Assay

Figure 1 shows typical qPCR traces generated with DNA Standards 1–4, a no template control, and a diluted Illumina library using either a Bio-Rad CFX96 Touch™ (top) or Applied Biosystems 7500 Fast with ROX normalization (bottom). The DNA library was diluted 1:100,000 as described in the protocol above. In these examples, the qPCR trace generated by the diluted library fell within the range of the qPCR traces from the DNA standards. The NTC was well separated from the last DNA standard (Standard 4) and the efficiency was calculated to be 94.4% (top) or 95.9% (bottom). The concentration of the example library was determined using the measured standard curves (right), adjusted by a size normalization factor (standard size divided by library size as measured by BioAnalyzer; 399/310 base pairs), and multiplied by the dilution factor (100,000) to give an undiluted library stock concentration of 69.8 nM (top) or 69.6 nM (bottom). For both instruments and software, default thresholds and calculation methods were used.

Figure 1a: Bio-Rad CFX96 Touch; Linear amplification plot, No ROX normalization

Figure 1b: Applied Biosystems 7500 Fast; Log amplification plot, ROX

Typical results from the NEBNext Library Quant Kit on a Bio-Rad CFX96 Touch (Figure 1a) and an Applied Biosystems 7500 Fast real-time qPCR instrument (Figure 1b). Amplification curves are shown on the left and resulting standard curves on the right. All default settings were used on both platforms. ABI 7500 Fast assay included 1X
ROX (Low Concentration) and the ROX normalization.