QuantStudio
™
3D Digital PCR System
Catalog Number
4482593 and 4481097
Pub. No.
MAN0008159
Rev.
B.0
Note:
For safety and biohazard guidelines, refer to the “Safety”
appendix in the QuantStudio
™
3D Digital PCR System User Guide
(Pub. no. MAN0007720). Read the Safety Data Sheets (SDSs) and
follow the handling instructions. Wear appropriate protective
eyewear, clothing, and gloves.
Operational workflow
The following figure shows the workflow for performing a single
experiment on the QuantStudio
™
3D Digital PCR System. The
procedures for sample and/or consumable preparation and result
analysis can vary depending on the specific experiment that you
are performing.
Start
q
Set up the PCR reaction by mixing sample, master
mix, and assay(s).
q
Load the PCR reaction onto a QuantStudio
™
3D
Digital PCR 20K Chip, apply a lid, load the assembly
with immersion fluid, then seal the loading port.
q
Perform the PCR using the ProFlex
™
2x Flat PCR
System (or Dual Flat Block GeneAmp
®
PCR System
9700).
q
Read the Digital PCR 20K Chip using the
QuantStudio
™
3D Digital PCR Instrument.
q
Review the results on the QuantStudio
™
3D
Instrument touchscreen.
q
Store or discard the Digital PCR 20K Chip.
q
Analyze the data using the QuantStudio
™
3D
AnalysisSuite
™
Software.
q
Finish
Prepare the digital PCR reactions
Required materials
• Genomic or complementary DNA sample(s)
• TaqMan
®
Assay(s) for your experiment
• QuantStudio
™
3D Digital PCR Master Mix
• Gloves, powder-free, nitrile
• Laboratory-grade, lint-free wipes
• Microcentrifuge
• Permanent marker or pen
• Pipettes and 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
Prepare the reaction mix and samples
Note:
The volumes of the following example protocol have been
adjusted so that two chips are run for each sample.
Table 1
PCR Reaction Mix
Material
Volume
(μL)
[1]
Stock
Final
Digital PCR Master Mix, 2X
17.4
2X
1X
TaqMan
®
Assay, 20X
1.7
20X
1X
Diluted DNA
3.5
[2]
10 ng/μL 1 ng/μL
Water
12.2
—
—
Total volume (for 1 sample/2 chips)
34.8
—
—
[1]
Volumes include 20% excess to compensate for volume loss from pipetting.
[2]
The recommended DNA volume is based upon a human gDNA sample at
10 ng/µL concentration with the target sequence present at two copies per
diploid genome. This recommended volume will vary depending upon species,
sample type, and sample concentration. For best results, we recommend
adding sufficient DNA such that the concentration of target sequence is
between 200 and 2,000 copies/µL.
1.
Remove from storage and allow the QuantStudio
™
3D Digital
PCR Master Mix and TaqMan
®
Assay(s) to warm at room
temperature.
QUICK REFERENCE
For Research Use Only. Not for use in diagnostic procedures.