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Chapter 3 Prepare the digital PCR reaction and load the chips
3
Prepare the digital PCR reactions
Determine the
optimal dilution
when the
target is
unknown
Prepare the digital PCR reactions
Required
materials
Guidelines for
dPCR sample
preparation
22
If the target copy number per genome is unknown (e.g., for a locus of unknown
copies per genome or RNA of unknown expression level), we recommend that you
determine the optimal dilution by preparing a dilution series of each sample that
includes three to four data points above and below the expected digital range. This
will ensure that one of the data points is within the optimal digital range. If real-time
data is available for the assay and sample, use it to determine the start and end
points of the dilution series.
• Genomic or complementary DNA sample(s)
• Fluorescent-labeled quantitative PCR reagents (TaqMan
Green primers) for your experiment
™
• QuantStudio
3D Digital PCR Master Mix v2
• Gloves, powder-free, nitrile
• Low-lint polyester wipes (clean-room grade)
• Microcentrifuge
• Permanent marker or pen
• Calibrated pipettes and barrier tips, P10 to P1000
• Reaction tubes, DNase/RNase-free, non-stick or low-binding, 0.5-mL or 1.5-mL
• TE Buffer, 1X Molecular Biology Grade
• Vortex mixer
• Water, DNase-free, sterile-filtered
When preparing samples for dPCR amplification:
• Use a positive-displacement pipette or aerosol-resistant pipette tips, and follow
proper pipette-dispensing techniques to prevent aerosols.
• Wear clean gloves and a clean lab coat (not previously worn while handling
amplified PCR products or used during sample preparation).
• Change gloves whenever you suspect that they are contaminated.
• Maintain separate areas and dedicated equipment and supplies for pre- and
post-PCR activities.
• Never bring amplified products into the PCR setup area.
• Open and close all sample tubes carefully. Centrifuge tubes before opening.
• Keep reactions and components capped as much as possible.
• Clean lab benches and equipment periodically with 10% bleach solution. Use
™
DNAZap
PCR DNA Degradation Solutions (Cat. No. AM9890).
™
Assays or SYBR
QuantStudio
™
3D Digital PCR System User Guide
™
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