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Clc genomics workbench price5/2/2023 ![]() ![]() ![]() Chips are then read in the Quantstudio 3D chip reader to obtain raw fluorescent values (Life Technologies). Chips are then sealed and cycled on a GeneAmp PCR system 9700 with a flatblock attachment using the following conditions Stage 1, 96° C for 10 minutes, Stage 2, 60° C for 2 minutes then 98° C for 30 seconds, repeat Stage 2 39 times, Stage 3, 60° C for 2 minutes and an infinite 10° C hold. Reactions are loaded onto Quantstudio 3D digital PCR chips using the Quantstudio 3D chip loader according to manufacturer’s instructions (Life Technologies). Digital PCR is then performed according to manufacturer’s instructions by mixing 3.33 µl diluted template DNA (concentrations of DNA are first determined by performing test dilution series) with 16.5 µl Quantstudio 3D master mix, 3.33 µl TaqMan assay, and 9 µl water (32.16 µl ) (Life Technologies). Total sample DNA quantification is carried out using fluorescent-based PicoGreen assay (Life Technologies). For a complete description of the dPCR methods see Masser et al., Age 2016 38:323-333.Īssays for human, mouse and rat genomes are available and can be generated for any species of interest. Based on the count of fluorescent positive and negative wells and using a Poisson distribution, the number of target copies can be calculated per microliter. Those wells that were loaded with template are positive wells, while those wells that were filled without template are negative. Reactions are then cycled to end-point and fluorescence is read in each well. Template is diluted to a point where there is either 0 or 1 copies per well. The reactions are distributed across a chip with 20,000 856-pl wells. Total genomic DNA is isolated from tissue/cells and mixed with proper assay components. Nuclear genome counting in parallel as a surrogate for cell number allows for normalization to cell number.Ībsolute mtDNA quantitation dPCR workflow. Using digital PCR to ‘count’ mitochondrial genomes provides an absolute quantitation, allowing data to be compared study to study. ![]() Changes in mitochondrial genome number have been observed with aging but traditional techniques have been limited as they only provide a relative quantitation. ![]()
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