• Apply the chip lid and add Immersion Fluid immediately after loading each chip
to avoid evaporation.
• Load and seal chips in batches of up to 24 chips (the maximum number that can
be loaded onto one thermal cycler).
• If you do not intend to load an opened chip immediately, cover the chip using the
plastic plate included in the chip package to prevent contamination. If you plan
to keep the opened chip for longer than a day, the chip should be stored with
dessicant.
• Use all of the Immersion Fluid within 60 minutes of uncapping the syringe. After
a syringe is opened, you cannot recap it for later use.
• Thermal cycle the chips within 2 hours after loading them.
Prepare the DNA samples
We recommend the following best practices for the preparation of DNA template,
genomic DNA (gDNA) or complementary DNA (cDNA), for use in digital PCR (dPCR)
experiments. Because dPCR experiment strategy and methodology can vary
significantly, sample preparation and template quality must be assessed on an
individual basis.
Use gDNA or cDNA template that:
• Is extracted from the raw material that you are testing with an optimized
protocol; salting-out procedures and crude lysates are not recommended
• Does not contain PCR inhibitors
• Has an A
260/230
and A
260/280
ratio between 1.7 and 1.9
The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA
and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is
generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case,
it may indicate the presence of protein, phenol, or other contaminants that absorb
strongly at or near 280 nm.
The ratio of absorbance at 260 nm and 230 nm is used as a secondary measure of
nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than
the respective 260/280 values. Expected 260/230 values are commonly in the range
of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the
presence of contaminants that absorb at 230 nm.
The quantity of DNA template added to a dPCR reaction depends on the:
• Concentration of gDNA or cDNA present in each sample
• Expected number of copies of the target sequence present in the genome or
cDNA of your samples
Quality of DNA
Quantity of
DNA
Chapter 3
Prepare the digital PCR reaction and load the chips
Prepare the DNA samples
3
20
QuantStudio
™
3D Digital PCR System User Guide
