Bio-Rad ddPCR™ Supermix for Probes - Manual (Page 12)

Chapter 1 QX200 Droplet Digital PCR System
QX200 Droplet Reader and QuantaSoft Software Instruction Manual 4 |
1.3 Droplet Digital PCR Workflow
Droplet Digital PCR involves the following steps (4.5–5.5 hours for the complete workflow):
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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)
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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
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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
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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 reading or quantifying droplets and recovering 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
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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.
1.4 System Setup and General Operation Instructions
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Connect the QX200 droplet generator and QX200 droplet reader to a power source using the power cords
provided. Leave 10" (5 cm) clear space behind and 5" (2.5 cm) clear to the right and left of each instrument
for proper ventilation
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Connect the QX200 droplet reader to the laptop PC using the USB 2.0 cord provided. QuantaSoft software
is preinstalled on the laptop
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Power on the QX200 droplet reader using the switch at the back. The status indicator turns solid green to
indicate power is on. The QX200 droplet generator is powered on by plugging it into a power source
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The droplet reader requires droplet reader oil (catalog #186-3004). Please refer to Section 4.2.2 for
instructions on replacing droplet reader oil