Prior to droplet generation, nucleic acid samples (DNA or RNA) are prepared as they are for any real-time assay: using primers, fluorescent probes (TaqMan probes with FAM and HEX or VIC), and a proprietary supermix developed specifically for droplet generation. Step 0: Prepare PCR-Ready Samples prior to Starting ddPCR The steps are described in more detail below. The PCR-positive and PCR-negative droplets are counted to provide absolute quantification in digital form. After PCR on a thermal cycler, droplets from each sample are streamed in single file on the QX200 Droplet Reader to count positive and negative reactions.
SDS DROP DEAD GENERATOR
First, the QX200 Droplet Generator partitions samples into thousands of nanoliter-sized droplets. This 3-D animated video describes the principles and process of ddPCR using Bio-Rad's Droplet Digital PCR System.
The greater number of partitions yields higher accuracy
Superior partitioning - ddPCR technology yields 20,000 droplets per 20 µl sample, nearly two million partitioned PCR reactions in a 96-well plate, whereas chip-based digital PCR systems produce only hundreds or thousands of partitions.Lower equipment costs - the emulsion-based reaction system means that the PCR reactions can be performed in a standard thermal cycler without complex chips or microfluidics.Reduced consumable costs - reaction volumes are in the pico- to nanoliter ranges, reducing reagent use and the sample quantity required for each data point.Simplified quantification - neither calibration standards nor a reference (the ΔΔCq method) is required for absolute quantification.Removal of PCR bias - error rates are reduced by removing the amplification efficiency reliance of qPCR, enabling the detection of small (1.2-fold) differences.Increased signal-to-noise ratio - high-copy templates and background are diluted, effectively enriching template concentration in target-positive partitions, allowing for the sensitive detection of rare targets and enabling a ☑0% precision in quantification.Unparalleled precision - the massive sample partitioning afforded by ddPCR enables the reliable measurement of small fold differences in target DNA sequence copy numbers among samples.Absolute quantification - ddPCR technology provides an absolute count of target DNA copies per input sample without the need for running standard curves, making this technique ideal for measurements of target DNA, viral load analysis, and microbial quantification.The benefits of ddPCR technology include: This partitioning enables the measurement of thousands of independent amplification events within a single sample.ĭdPCR technology enables high-throughput digital PCR in a manner that uses lower sample and reagent volumes and reduces overall cost compared with other methods while maintaining the sensitivity and precision that are the hallmarks of digital PCR. In traditional PCR, a single sample offers only a single measurement, but in Droplet Digital PCR, the sample is partitioned into 20,000 nanoliter-sized droplets. Sample partitioning is the key to droplet digital PCR. This technique has a smaller sample requirement than other commercially available digital PCR systems, reducing cost and preserving precious samples. The Droplet Digital PCR System partitions nucleic acid samples into thousands of nanoliter-sized droplets, and PCR amplification is carried out within each droplet. The massive sample partitioning is a key aspect of the ddPCR technique. The droplets serve essentially the same function as individual test tubes or wells in a plate in which the PCR reaction takes place, albeit in a much smaller format. Droplets are formed in a water-oil emulsion to form the partitions that separate the template DNA molecules. Droplet Digital PCR technology is a digital PCR method utilizing a water-oil emulsion droplet system.
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