User manual

Droplet Digital
™
PCR Applications Guide | 19
Fig. 2.6. Labeling the plate.
After heat sealing, place the PCR plate in a thermal cycler for PCR using the
following guidelines.
■
Use a recommended thermal cycling protocol
■
Use a 2.5°C/sec ramp rate to ensure each droplet reaches the correct temperature
for each step during the cycling
■
40 cycles of PCR is enough for an optimized ddPCR assay. Do not exceed 50 cycles
■
After PCR, the plate can be left in the thermal cycler overnight at 10°C or stored
at 4°C. Do not store the plate for more than 3–4 days before running it in a QX100 or
QX200 droplet reader
Setting Up an Experiment in QuantaSoft
™
Software
From the computer attached to the droplet reader, open QuantaSoft software in the
setup mode and design a new plate with a layout according to your experimental design.
Detailed instructions for how to set up a new experiment and interpret ddPCR data can
be found in the user manual.
Double click on a well in the plate layout to open the Well Editor dialog box. Designate the
sample name, experiment type, and which assays correspond to which channels, such
as FAM and HEX (Figure 2.6). You can select several contiguous wells at one time using
shift + double click or select non-contiguous wells using Ctrl + double click. Either selection
will bring up the labeling menu. In the Well Editor dialog box, input sample names and use
the dropdown menu to designate the experiment type.
There are three types of experiments that can be selected for each well:
■
ABS — absolute quantification
■
RED — rare target sequence detection (rare event detection)
■
CNV — copy number variation to measure the concentration of target relative
to the concentration of a reference
Select Apply to load the wells and when finished select OK. Once the plate layout is
complete, select Run to begin the droplet reading process.
Designing Droplet Digital
™
PCR Experiments