User guide

Prepare Samples and Load
Reactions
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Prepare the DNA samples ............................................. 36
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Prepare the digital PCR reactions ....................................... 38
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Load the Digital PCR 20K Chips ........................................ 40
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Load the chips using the Chip Loader .................................. 42
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Load chips manually ................................................. 50
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.
To ensure high quality data, we recommend that the gDNA or cDNA template you
use for dPCR experiments:
• 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
Note: 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
2
Quality of DNA
Quantity of DNA
36 QuantStudio
®
3D Digital PCR System User Guide