QIAcuity EvaGreen (EG) PCR Kit

If you had analyzed your nanoplates without VFP, the impact depends on the variation of the partition volume of the new nanoplate batch compared to the latest. The VPF reduces the CV from approximately 5% to 2%. We recommend to reanalyze results in case the data originated from different wells (e.g., copy number variations or gene expression data sets for which the reference sample was measured in a different well). Results obtained across different plates should also be r-analyzed. A reanalysis is not required for assay data that were analyzed within the same well (e.g., mutation rate determination using two channels within the same well).

A standard PCR plate is required to set up dPCR reaction before transferring it to the nanoplate to ensure a proper mixing of the reaction mix before partitioning. 

QIAGEN master mixes are optimized for nanoplate microfluidics and are recommended to be used with our dPCR system. They also include an optimized reference dye required for proper analysis.

All DNA samples used in reaction mixes should show similar quality and quantity, which can easily be assessed using UV spectrophotometry. DNA samples with an average length of ≥20 kb (e.g., genomic DNA purified via spin column with silica membrane) should be fragmented by restriction digestion before partitioning. Enzymatic fragmentation of larger DNA ensures an even distribution of template throughout the QIAcuity Nanoplate, which in turn leads to an accurate and precise quantification.

The QIAcuity Probe PCR Kit should be stored immediately upon receipt at –30 to –15°C in a constant-temperature freezer and protected from light. The QIAcuity Probe PCR master mix can also be stored protected from light at 2–8°C. Components are stable for 12 months, unless otherwise indicated on the label. 
The QIAcuity EG PCR Kit should be stored immediately upon receipt at –30 to –15°C in a constant-temperature freezer and protected from light. The QIAcuity EG PCR master mix can also be stored protected from light at 2–8°C. Components are stable for 6 months, unless otherwise indicated on the label. 
The QIAcuity Nanoplates does not have expiry date and are stable for at least 1 year when stored at RT. 

The plate is designed for a single use run. For example, even if only 30 samples are loaded into the 96-well plate, a whole plate will be sealed by the roller. It can't be unsealed and used for another run. The QIAcuity Software won’t allow to set up a separate experiment for the same nanoplate to avoid that previously processed plates being not partitioned a second time.

An essential temperature gradient functionality was introduced with software version 2.5. When updating older software versions to 2.5, each QIAcuity instrument will offer the temperature gradient and may be used to find the best cycling temperature for new dPCR assays. When running a QIAcuity Four or a QIAcuity Eight, all plates may have their own temperature profile, including the option for a temperature gradient.

The VPF provides a set of well-specific and molding form-specific factors used to specify the exact reaction volume of a nanoplate, thus increasing the concentration calculation of each well. 

 In general, nanoplates provide partitions of fixed sizes that enable a very precise way of sample concentration calculation. If a new molding form is used for nanoplate manufacturing, potential variation of partition sizes can be addressed by applying the molding form-specific VPF. Thus, each time a new molding form is used, a new VPF is created and made available. Currently, the VPF is updated once every 3–6 months.

In dPCR we measure the absolute concentration of targets at endpoint reaction. Concentrations of unknowns can be determined based on dPCR results observed (number of negatives, number of positives, and partition volume analyzed).