Get the most out of your enzyme with our documents, guides, brochures and tools
Browse our frequently asked technical questions by selecting a product below.
Examine the melt curve to ensure that there is a single peak. If you see multiple peaks, there may be contamination present or unligated adapters. Otherwise, if the samples appear before DNA Standard 1, dilute the sample 100x and repeat. If the samples appear after DNA Standard 5, use a sample that is 100x more concentrated.
If the overall efficiency of your standard curve is between 90% – 110% and your R2 value is close to 1, this is not a cause for concern. As long as the slope of the line through all of the standards is between 3.1 – 3.6, the quantification will be accurate.
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.
If the peak overlaps with the shoulder seen in the standards’ melting peaks, this is likely amplification of the Illumina® primers. This can occur when the primer/template ratio is too high, or when there are large amounts of unligated adapters. If the peak is significant, the quantification results are not reliable and the experiment should be repeated. Ensure that the correct amount of primer is added to the master mix and use a higher concentration of template if possible. If the problem persists, the library may need to go through size selection again to remove free adapters.
If multiple peaks are observed that do not correspond to the adapter peak, the most likely causes are either contamination or unsuccessful size selection during library preparation. Repeat the measurement with fresh dilutions of the library. If the problem persists, that library may need to undergo further size selection.
It is normal to see a slight shoulder on the low-temperature side of the sample melt peak. If the shoulder is very large, either extra Illumina® Primer mix was added or an insufficient amount of standard was added. In this case, the experiment should be repeated with special care taken while making the master mix and when adding standards to the reaction.
If multiple peaks are observed in the melt curves of the standards, then contamination was introduced into the reaction. If it is present in all of the standards, the contamination is in the master mix. The experiment should be repeated with new aliquots of reagents. If it is present in the replicates of only one standard, that standard aliquot has become contaminated and must be discarded.
If the R2 value is not close to 1, this suggests that the standards are not spaced evenly. Inspect the standard curve chart and remove any points that are far from the line. As long as there are three standards with two measurements each, the quantification will be accurate.
If you are unable to bring the quality of the fit to the necessary level, the experiment should be repeated. Take extra care when pipetting the standards to ensure accurate volumes and prevent contamination.
What if the measurement I get with my NGSBIO Library Quant Kit does not agree with my Qubit, spectrophotometer, or another method measurement?
Instruments like the Qubit or a spectrophotometer measure the total amount of DNA present in solution. The same is true of concentration measurements by an Agilent Bioanalyser. The NGSBIO Library Quant Kit for Illumina® will only quantify molecules with both adapters present. In addition, qPCR is a much more accurate method in general for quantifying DNA compared to fluorescent or spectroscopic methods. It is normal for these different measurement methods to disagree.
If the concentration measured by qPCR is significantly lower than what is measured by another method, this suggests that only a small proportion of molecules correctly incorporated the adapters during adapter ligation.
Check the melt curves to ensure there is a single product peak. If there are multiple peaks, this suggests the sample may have become contaminated or degraded, or that an error occurred during the size selection or adapter ligation steps or library preparation. If the melt analysis shows only one peak, the variation is probably caused by inaccurate pipetting. Create a new dilution of the sample and repeat the experiment, taking care to pipette the replicates accurately. If the problem persists, a new library may need to be created.
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.