Thermo Scientific Applied Biosystems QuantStudio - Determine the Optimal Dilution When the Target Is Unknown; Prepare the Digital PCR Reactions; Required Materials; Guidelines for Dpcr Sample Preparation

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.
Prepare the digital PCR reactions
•
Genomic or complementary DNA sample(s)
•
Fluorescent-labeled quantitative PCR reagents (TaqMan
™
Assays or SYBR
™
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-ltered
When preparing samples for dPCR amplication:
•
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
amplied 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 amplied 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).
Determine the
optimal dilution
when the
target is
unknown
Required
materials
Guidelines for
dPCR sample
preparation
Chapter 3 Prepare the digital PCR reaction and load the chips
Prepare the digital PCR reactions
3
22
QuantStudio
™
3D Digital PCR System User Guide