User manual

20 | Droplet Digital
™
PCR Applications Guide
Droplet Reading
Following PCR amplification of the nucleic acid target in the droplets, place the PCR plate
in a QX100 or QX200 droplet reader. The droplet reader and QuantaSoft software count the
PCR-positive and PCR-negative droplets to provide absolute quantification of target DNA.
Droplet reading considerations are as follows:
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Before a run, the instrument can be set to interrogate droplets either in rows or columns
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Ensure there is enough droplet reader oil in the instrument and the waste is empty before
a run
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Each sample is processed individually and interrogated for both FAM and HEX (or VIC)
fluorescence
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Data from 12,000–16,000 droplets are used in concentration calculations
■
The reader measures fluorescence intensity of each droplet and detects the size
and shape as droplets pass the detector; droplets are excluded if they do not meet
quality metrics
Data Analysis
After the QX100 or QX200 droplet reader has finished interrogating all wells, use QuantaSoft
software to analyze the data in each well. It automatically opens the first well with data to
begin the analysis. If the plate was set up for ABS analysis, automatic thresholding determines
concentrations and populates the data tables in the analysis mode of the software.
Important: The concentration reported is copies/µl of the final 1x ddPCR reaction.
Well data must meet certain quality metrics before QuantSoft software will automatically
calculate a threshold above which droplets are considered positive. The threshold may be
manually adjusted on a well-by-well basis or across an entire plate (Figure 2.7).
Designing Droplet Digital
™
PCR Experiments