User guide

If the target copy number per genome is unknown, say for a locus of unknown copies
per genome or RNA of unknown expression level, we recommend that you determine
the optimal dilution by loading and imaging a dilution series of each sample at the
expected digital range. By assaying three to four data points above and below the
expected digital range, you ensure that one of the data points is within the optimal
digital range. Should real-time data be available for the assay and sample being used,
this can guide the starting and end point of the dilution series.
Prepare the digital PCR reactions
The following materials are required to prepare digital PCR reactions for use on the
QuantStudio
®
3D Digital PCR System.
•
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
• Gloves, powder-free, nitrile
• Cleanroom-grade, low-lint polyester wipes
• 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
• Vortexer
• Water, DNase-free, sterile-filtered
When preparing samples for PCR amplification:
• Use a positive-displacement pipette or aerosol-resistant pipette tips.
• 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:
– Sample preparation
– PCR setup
– PCR amplification
– Analysis of PCR products
• Never bring amplified PCR products into the PCR setup area.
• Open and close all sample tubes carefully. Centrifuge tubes before opening.
Try not to splash or spray PCR samples.
• Keep reactions and components capped as much as possible.
• Clean lab benches and equipment periodically with 10% bleach solution. Use
DNAZap
™
Solution (PN AM9890).
Determine the
optimal dilution
when the target is
unknown
Required
materials
Guidelines for
PCR sample
preparation
Chapter 2 Prepare Samples and Load Reactions
Prepare the digital PCR reactions
2
38 QuantStudio
®
3D Digital PCR System User Guide