Bio-Rad’s QX200 ddPCR system combines water-oil emulsion droplet technology with microfluidics. The QX200 droplet generator partitions samples into 20,000 droplets. PCR amplification is carried out within each droplet using a thermal cycler. After PCR, droplets are streamed in single file on a QX200 droplet reader, which counts the fluorescent positive and negative droplets to calculate target DNA concentration.
- Unparalleled precision — the massive sample partitioning afforded by ddPCR enables small fold differences in target DNA sequence between samples to be reliably measured
- Increased signal-to-noise — enrich for rare targets by reducing competition that comes from high-copy templates
- Removal of PCR efficiency bias — error rates are reduced by removing the amplification efficiency reliance of PCR, enabling accurate quantification of targets
- Simplified quantification — a standard curve is not required for absolute quantification
A video introduction of Droplet Digital PCR
Droplet Digital PCR Workflow
Droplet Digital PCR involves the following steps (4.5–5.5 hours for the complete workflow):
- Prepare PCR-ready samples — combine nucleic acid sample (DNA or RNA), primers, and probes (FAM, VIC, or HEX) or intercalating dye (EvaGreen) with Bio-Rad ddPCR supermix (see Table 1.2)
- Make droplets — load 20 μl of the ddPCR reaction into the DG8 droplet generator cartridge, then load the cartridge into the QX200 droplet generator to partition the sample into droplets. The QX200 droplet generator uses microfluidics to combine oil and aqueous sample to generate the nanoliter-sized droplets required for ddPCR analysis. It processes up to eight samples at a time in about 2 minutes
- Perform PCR — pipet droplets from the cartridge to a 96-well PCR plate and seal the plate with foil using a PX1 plate sealer (see Table 1.2). Perform PCR to end point (~40 cycles) using a thermal cycler
- Read droplets — load the plate into the QX200 droplet reader and start your run. The droplet reader sips each sample, singulates the droplets, and streams them in single file past a two-color detector. The detector reads the droplets to determine which contain a target (+) and which do not (–). If the goal is to read or quantify droplets and recover material from droplets in parallel, prepare two sets of reactions, one for each application. For example, a set of eight wells in a single DG8 cartridge can be generated: four of these will be read after thermal cycling, and four will not be read.
- Analyze results — the droplet reader connects to a laptop computer running QuantaSoft software. The software provides a complete set of tools for setting up and naming samples, running and controlling the instrument, and analyzing results. It also reads the positive and negative droplets in each sample and plots the fluorescence droplet by droplet. The fraction of positive droplets in a sample determines the concentration of target in copies/μl
The QX200 ddPCR system is compatible with hydrolysis probe (TaqMan) chemistry and can detect up to two probes at a time (FAM/VIC or FAM/HEX), using a dye deconvolution matrix in the software to ensure target specificity. It is also compatible with EvaGreen chemistry. Use only the approved Bio-Rad supermixes listed in Table 1.2 with this system; using unapproved supermixes may harm the instrument and voids the warranty.
