Geode H14000 User Manual
14
•
it could be useful to aliquot some reagents to prevent contamination and, at the same time, to
reduce repetitive freezing/thaw cycles that might affect their efficiency.
Protocol for setting up
Crystal Digital PCR™
a) Sample purification and pre-treatment
A range of extraction kits and methods can be used to purify nucleic acids from a variety of
sample types, However, individual sample-type compatibility for digital PCR applications may
require a dedicated assay validation by the end-
user. DNA samples with ≥10 kb average length
(e.g., genomic DNA) should be fragmented by restriction digestion before partitioning to ensure
even distribution of the DNA template during partitioning. Restriction digestion is not required for
highly fragmented DNA (e.g., FFPE DNA or circulating DNA). Care must be taken to use
restriction enzymes that do not cut within the amplified sequence.
Reaction mix preparation
For Cryst
al Digital PCR™, Stilla Technologies recommends using the naica® PCR MIX reagents
https://www.stillatechnologies.com/naica-system-dpcr-mixes/
Always thaw each reagent, vortex, and spin briefly in a microcentrifuge to collect the material in
the bottom of the tube.
i.
Fluorescently labeled probes
When using fluorescently labeled TaqMan® probes, Stilla Technologies recommends
using the naica® multiplex PCR MIX.
For the reaction mix composition using the naica® multiplex PCR MIX, view the detailed reaction
preparation
information
in
the
corresponding
IFU
here:
https://www.stillatechnologies.com/technical-resources/naica-system-prism3/
Please refer to the next section for guidelines on primer and probe concentrations.
For other compatible PCR Master mix compositions, refer to the IFU for the Quantabio reagents
for
the
naica®
system
available
here:
https://www.stillatechnologies.com/technical-
resources/naica-system-prism3/
Vortex and centrifuge the reaction mix after adding the reagents to avoid air bubbles that may
compromise the
Crystal Digital PCR™ reaction.
ii.
Non-specific fluorescent intercalating dye: EvaGreen®
When using EvaGreen®, Stilla Technologies recommends using the naica® PCR MIX.
For the reaction mix composition using the naica® PCR MIX, view the detailed reaction
preparation information in the corresponding IFU here:
https://www.stillatechnologies.com/technical-resources/naica-system-prism3/
Optimization of
Crystal Digital PCR™
i.
Fluorescently labeled probes
The optimal primer and probe concentrations as well as the optimal hybridization/elongation
temperature during the cycling should notably be tested. Optimal results involve good separability
between positive and negative droplets, no or few
“rain” droplets (positive droplets with variable
intensity localized between the negative and positive cluster) that could be due for example to
