Thermo Scientific Applied Biosystems QuantStudio User manual

For Research Use Only. Not for use in diagnostic procedures.
QuantStudio™3D Digital PCR System
USER GUIDE
for use with:
QuantStudio™3D Digital PCR Instrument
ProFlex™2 × Flat PCR System or Dual Flat Block GeneAmp™PCR
System 9700
QuantStudio™3D Digital PCR 20K Chip v2
QuantStudio™3D Digital PCR Master Mix v2
QuantStudio™3D Digital PCR Chip Loader
Catalog Number A29154
Publication Number MAN0007720
Revision E.0

Manufacturer:
Life Technologies Holdings Pte Ltd |
Block 33 |
Marsiling Industrial Estate Road 3 |
#07-06, Singapore 739256
Products:
QuantStudio™3D Digital PCR Instrument
ProFlex™2 × Flat PCR System
Dual Flat Block GeneAmp™PCR System 9700
QuantStudio™3D Digital PCR Chip Loader
Manufacturer:
Life Technologies Corporation |
6055 Sunol Blvd |
Pleasanton, CA 94566
Products:
QuantStudio™3D Digital PCR 20K Chip v2
Life Technologies Corporation |
7335 Executive Way | Frederick, MD
21704 | USA
Products:
QuantStudio™3D Digital PCR Master Mix v2
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0007720
Revision Date Description
E.0 18 March 2020 Removed the v1 chip and master mix. Updated template and legal
information. Updated “Prepare the dPCR reaction mix for each sample”
on page 23. Updated thermal cycling parameters. Reformatted “Install
the GeneAmp™PCR System 9700” on page 73.
D.0 15 July 2015 Updated for v2 chips, lids, and master mix. Also updated with changes
to instrument rmware, including adjusting for the different well volumes
between v1 and v2 chips. Includes improvements to the chip loading and
imaging workows.
C.0 February 2015 Updated for rmware and software revisions, including changes to
instrument networking, thermal cycling, chip analysis, maintenance,
software access, and computer requirements. Updated to latest
corporate boilerplate. Updated general sample and chip preparation,
system installation, troubleshooting, and parts and materials.
B.0 April 2014 Added support for the ProFlex™PCR System and revised the chip
loading instructions.
A.0 December 2013 Updated the manual chip preparation and added procedures for chip
preparation using the QuantStudio™3D Digital PCR Chip Loader and
wireless network installation.
02 June 2013 Updated general chip preparation and instrument networking.
01 March 2013 Initial version
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a
registered trademark of Roche Molecular Systems, Inc., used under permission and license.
©2020 Thermo Fisher Scientific Inc. All rights reserved.

Contents
■CHAPTER 1 Product information ....................................... 8
About the QuantStudio™3D Digital PCR System .................................... 8
Instruments, kits, consumables, and accessories ................................... 9
Required materials not supplied ................................................. 10
Power line regulator ........................................................ 12
Surge protector ............................................................ 12
About the instrument ........................................................... 12
Data collection ............................................................ 13
Supported dyes ........................................................... 13
About the software ............................................................. 13
System requirements ....................................................... 13
About the chip ................................................................. 14
■CHAPTER 2 Getting Started ........................................... 15
Operational workow ........................................................... 16
Connect the QuantStudio™3D Digital PCR Instrument to a network .................. 17
Connect the QuantStudio™3D AnalysisSuite™Software ............................ 17
Choosing a thermal cycler ....................................................... 17
Compatible thermal cyclers ................................................. 17
■CHAPTER 3 Prepare the digital PCR reaction and load
the chips .................................................................. 19
Guidelines for loading and sealing chips .......................................... 19
Prepare the DNA samples ....................................................... 20
Quality of DNA ............................................................ 20
Quantity of DNA ........................................................... 20
Sample dilution ............................................................ 21
QuantStudio™3D Digital PCR System User Guide 3

Determine the optimal dilution when the target is known ........................ 21
Determine the optimal dilution when the target is unknown ...................... 22
Prepare the digital PCR reactions ................................................ 22
Required materials ......................................................... 22
Guidelines for dPCR sample preparation ..................................... 22
Recommended DNA volume and concentration ............................... 23
Prepare the dPCR reaction mix for each sample ............................... 23
Load the chips using the QuantStudio™3D Digital PCR Chip Loader ................. 24
Materials required ......................................................... 24
Chip loader status light ..................................................... 25
Prepare the chip loading workspace ......................................... 25
Prepare the syringe containing the Immersion Fluid ............................ 26
Load and seal the chips .................................................... 27
Fill the chip case with Immersion Fluid ....................................... 30
Seal the chip case with the label ............................................. 31
■CHAPTER 4 Thermal cycle the chips ................................. 32
Materials required .............................................................. 32
Prepare the thermal cycler ...................................................... 32
Guidelines for handling loaded chips ............................................. 35
Perform the PCR ............................................................... 36
Unload the thermal cycler ....................................................... 38
■CHAPTER 5 Image and analyze the chips ........................... 40
About imaging and primary analysis .............................................. 40
QuantStudio™3D System data ow .......................................... 40
About the instrument interface ................................................... 41
Chip imaging .................................................................. 42
Materials required ......................................................... 42
Guidelines for imaging multiple chips ......................................... 42
Specify the data destination ................................................. 42
Image the chips ........................................................... 43
Primary analysis ............................................................... 46
About imaging data and primary analysis ..................................... 46
About the results screen .................................................... 47
Data quality ags .......................................................... 48
About data les ............................................................ 48
Transfer the results ......................................................... 49
Using the QuantStudio™3D AnalysisSuite™Software .............................. 51
Access the software from a Connect cloud-based platform account ............. 51
Access the software from the server ......................................... 51
Obtain information from the Help system ..................................... 53
Contents
4QuantStudio™3D Digital PCR System User Guide

■CHAPTER 6 Troubleshooting .......................................... 54
Troubleshooting chip images using the Chip View .................................. 54
■APPENDIX A Install the QuantStudio™3D Digital PCR System .... 61
Time required for installation ..................................................... 61
Materials required .............................................................. 61
Before you begin ............................................................... 61
Plan the laboratory layout ....................................................... 62
Choose the instrument locations ................................................. 62
Install the QuantStudio™3D Digital PCR Instrument ................................ 63
Install the ProFlex™2 × Flat PCR System ......................................... 68
Unpack and place the ProFlex™PCR System Base Module ..................... 68
Prepare the workspace and instrument ....................................... 69
Install the Tilt Base ......................................................... 69
Unpack and install the sample block module .................................. 71
Set up the ProFlex™PCR System ............................................ 71
Install the GeneAmp™PCR System 9700 .......................................... 73
Unpack and place the GeneAmp™PCR System 9700 Base Module .............. 73
Unpack and install the Dual Flat Block Module ................................ 74
Install the Tilt Base and Chip Adapter ........................................ 75
Set up the GeneAmp™PCR System 9700 ..................................... 75
■APPENDIX B Specications and Layout ............................. 79
Component dimensions and weights ............................................. 79
QuantStudio™3D Digital PCR Instrument layout and connections .................... 80
Instrument clearances .......................................................... 80
Electrical specications ......................................................... 81
Environmental requirements ..................................................... 82
Network requirements .......................................................... 83
■APPENDIX C Maintenance ............................................ 84
Cleaning the chip tray and sample block .......................................... 84
Materials required ......................................................... 84
Clean the chip tray or sample block .......................................... 85
Congure the instrument settings ................................................ 86
(Optional) Set the instrument name .......................................... 86
Set the date and time ...................................................... 87
Check the instrument rmware .................................................. 87
Update the instrument rmware ............................................. 88
Returning your instrument for service ............................................. 89
Contents
QuantStudio™3D Digital PCR System User Guide 5

Replace the instrument fuses .................................................... 90
Materials required ......................................................... 90
Replace the fuses .......................................................... 90
Calibrate the touchscreen ....................................................... 91
View the instrument log ......................................................... 92
■APPENDIX D Networking .............................................. 94
Networking overview ........................................................... 94
About the network port and wireless adaptor .................................. 94
Networking guidelines and best practices ..................................... 95
Connect the instrument to the network ........................................... 95
Network setup workow .................................................... 96
Materials required ......................................................... 96
Required network information ............................................... 96
Connect the QuantStudio™3D Digital PCR Instrument to a network .............. 98
Create a network destination to receive run data ............................. 103
■APPENDIX E Load chips manually .................................. 106
Guidelines for loading and sealing chips ......................................... 106
Materials required ............................................................. 107
Prepare the syringe containing the Immersion Fluid ............................... 107
Load and seal the chips manually ............................................... 108
Fill the chip case with Immersion Fluid ........................................... 111
Seal the chip case with the label ................................................ 112
■APPENDIX F Safety ................................................... 113
Symbols on this instrument .................................................... 113
Conformity symbols ...................................................... 114
Safety alerts on this instrument ................................................. 115
Location of safety alerts on the instrument ................................... 115
Safety information for instruments not manufactured by Thermo Fisher Scientic ..... 116
Instrument safety ............................................................. 116
General ................................................................. 116
Electrical safety .......................................................... 117
Cleaning and decontamination ............................................. 118
Safety and electromagnetic compatibility (EMC) standards ......................... 118
Safety ................................................................... 119
EMC .................................................................... 119
Environmental design ..................................................... 119
Chemical safety .............................................................. 120
Biological hazard safety ....................................................... 122
Contents
6QuantStudio™3D Digital PCR System User Guide

■Documentation and support ........................................... 123
Related documentation ........................................................ 123
Customer and technical support ................................................ 123
Limited product warranty ...................................................... 124
Contents
QuantStudio™3D Digital PCR System User Guide 7

Product information
About the QuantStudio™3D Digital PCR System
The QuantStudio™3D Digital PCR System provides quantitative and qualitative
detection of target nucleic acid sequences (targets) using post-PCR (endpoint)
analysis.
Use the QuantStudio™3D Digital PCR Instrument to perform imaging and preliminary
analysis of a QuantStudio™3D Digital PCR 20K Chip v2. The chip must have been
loaded with uorescent-labeled quantitative PCR reagents (TaqMan™probe-based
assays or SYBR™Green primer-based assays) and thermal cycled using a ProFlex™
2 × Flat PCR System (or Dual Flat Block GeneAmp™PCR System 9700). The
QuantStudio™3D AnalysisSuite™Software performs subsequent analysis and post-
processing to yield relative or absolute quantication results from the raw imaging
data.
1
4
3
6
7
8
59
10
212
11
Figure 1 Components of the QuantStudio™3D Digital PCR System
1QuantStudio™3D Digital PCR Thermal Pads
2QuantStudio™3D Digital PCR Chip Adapters
3ProFlex™2 × Flat PCR System (or Dual Flat Block GeneAmp™PCR System 9700)
4QuantStudio™3D Tilt Base
5QuantStudio™3D Digital PCR Instrument
6QuantStudio™3D Digital PCR Chip Lid v2
7QuantStudio™3D Digital PCR Sample Loading Blade
8QuantStudio™3D Digital PCR 20K Chip v2
9(Optional) QuantStudio™3D Digital PCR Chip Loader
10 Immersion Fluid syringe
11 QuantStudio™3D Digital PCR Master Mix v2
12 Fluorescent-labeled quantitative PCR reagents (TaqMan™Assays or SYBR™Green primers)
13 (Not shown) QuantStudio™3D AnalysisSuite™Software
1
8QuantStudio™3D Digital PCR System User Guide

Instruments, kits, consumables, and accessories
The following tables describe the products covered in this user guide.
Instrument system
Item Catalog No. Amount
QuantStudio™3D Digital PCR System package
with v2 chips and master mix, includes:
•QuantStudio™3D Digital PCR Instrument
•QuantStudio™3D Digital PCR Chip Loader
•QuantStudio™3D Digital PCR 20K Chips v2
•QuantStudio™3D Digital PCR Master Mix
v2
•ProFlex™2 × Flat PCR System
A29154 1 system
QuantStudio™3D Digital PCR Instrument with
power cord
4489084 1 instrument
QuantStudio™3D Digital PCR Tilt Base for
ProFlex™Thermal Cycler
A24898 1 base
QuantStudio™3D Digital PCR Tilt Base for
GeneAmp™PCR System 9700
4486414 1 base
QuantStudio™3D Digital PCR Chip Adapter Kit
for Flat Block Thermal Cycler
4485513 1 adapter
QuantStudio™3D Extended Arm WiFi Adapter A28598 1 wireless adapter
QuantStudio™3D Digital PCR 20K Chip Kit v2
and Master Mix v2, includes:
•8 × 12 packs of v2 chips, chip lids, and
QuantStudio™3D Digital PCR Sample
Loading Blades
•24 syringes of Immersion Fluid, plus tips
•1.5 mL of QuantStudio™3D Digital PCR
Master Mix v2
A26317 1 kit (96 chips)
QuantStudio™3D Digital PCR 20K Chip Kit v2,
includes:
•12 v2 chips, chip lids, and
QuantStudio™3D Digital PCR Sample
Loading Blades
•3 syringes of Immersion Fluid, plus tips
A26316 1 kit (12 chips)
QuantStudio™3D Digital PCR Immersion Fluid,
1.6 mL per syringe
A27322 3 syringes
Chapter 1 Product information
Instruments, kits, consumables, and accessories 1
QuantStudio™3D Digital PCR System User Guide 9

Instrument system
Item Catalog No. Amount
QuantStudio™3D Digital PCR Chip Loader 4482592 1 loader
QuantStudio™3D Digital PCR Chip Lids v2 and
QuantStudio™3D Digital PCR Sample Loading
Blades
A29247 12 each
QuantStudio™3D Digital PCR Master Mix v2 A26358
A26359
1.5 mL
5.0 mL
QuantStudio™3D Digital PCR Reagent Kit,
includes:
•Assays and reagents for system installation
and verication, or training of operators on
the use of the system
A26360 1 kit
QuantStudio™3D Digital PCR Starter Kit,
includes:
•QuantStudio™3D Digital PCR Reagent Kit
• QuantStudio™3D Digital PCR
20K Chip Kit v2 (12 chips)
A26361 1 kit
Required materials not supplied
This system uses common molecular biology equipment, supplies, and reagents.
Unless otherwise indicated, all materials are available through thermofisher.com.
"MLS" indicates that the material is available from fisherscientific.com or another
major laboratory supplier.
Item Source
Clean room-grade, low-lint polyester wipes Fisher Scientic
(Cat. No. NC0375992) or MLS
Gloves, powder-free, nitrile MLS
Microcentrifuge MLS
Pipette tips, 10 to 1000 μL MLS
Pipettes, P10 to P1000 MLS
Reaction tubes, DNase/RNase-free, non-stick or
low-binding: 0.5-mL or 1.5-mL
Fisher Scientic
(Cat. No. 13-864-253 or
13-864-254) or MLS
Vortexer MLS
Chapter 1 Product information
Required materials not supplied
1
10 QuantStudio™3D Digital PCR System User Guide

Item Source
Water, DNase-free, sterile-ltered MLS
Water, deionized MLS
Isopropanol MLS
(Required for manual chip loading only) Heated
block (capable of maintaining 40±1°C)
MLS
Chapter 1 Product information
Required materials not supplied 1
QuantStudio™3D Digital PCR System User Guide 11

Use a 1.5-kVA power line regulator in areas where the supplied power uctuates in
excess of ± 10% of the normal voltage. Power uctuations can adversely affect the
function of the systems.
Note: A power line regulator monitors the input current and adjusts the power
supplied to the systems. It does not protect against a power surge or failure.
Use a 10-kVA surge protector (line conditioner) in areas with frequent electrical
storms or near devices that are electrically noisy, such as refrigerators, air
conditioners, or centrifuges. Short-duration, high-voltage power uctuations can
abruptly terminate the function of, and thereby damage the components of, the
systems.
Note: A dedicated line and ground between the systems and the building’s main
electrical service can also prevent problems caused by power uctuations.
About the instrument
The QuantStudio™3D Digital PCR Instrument consists of the components shown in
the following gure.
1
2
3
4
5
6
7
8
1Touchscreen—Provides access to the instrument functions, such as data transfer and
instrument operation.
2Chip tray—Conveys the QuantStudio™3D Digital PCR 20K Chip v2 to and from the imaging
stage in the interior of the instrument.
3USB port—Provides USB communication with the instrument. Can be used to transfer data to
and from the instrument.
4Fuse cover—Dual 1.6A, Time Lag T, 250 VAC, 5 × 20-mm electrical fuses that protect the
instrument from excessive electrical current.
5Power switch—Power switch for the instrument, where the states are on ( | ) or off ( O ).
6Power port—A 100–240 VAC port that provides power to the instrument.
7Ethernet port—An RJ45 port that provides Ethernet (gigabit) communication with the
instrument.
Power line
regulator
Surge
protector
Chapter 1 Product information
About the instrument
1
12 QuantStudio™3D Digital PCR System User Guide

The QuantStudio™3D Digital PCR Instrument collects raw uorescence data from the
QuantStudio™3D Digital PCR 20K Chip v2 following PCR amplication.
The instrument performs multiple image captures of the chip in the following three
phases:
1. Excitation – The instrument illuminates all wells of the chip, exciting the
uorophores in each reaction.
2. Emission Collection – The instrument optics collect the residual uorescence
emitted from the wells. The resulting image consists of light corresponding to
the range of emission wavelengths for the lter(s) in use.
3. Interpretation – The instrument assembles a digital representation of the
residual uorescence collected over a xed time interval.
After a run, the instrument determines the location and intensity of the uorescent
signals in each image, the dye associated with each uorescent signal, and the
signicance of the signal.
The QuantStudio™3D Digital PCR System features a lter set that is optimized for the
Applied Biosystems™FAM™, ROX™, and VIC™dyes.
About the software
The QuantStudio™3D Digital PCR System includes a software suite used to analyze
and manage digital PCR data generated by the QuantStudio™3D Digital PCR
Instrument. The QuantStudio™3D AnalysisSuite™Software includes a server and
Connect cloud-based platform deployment options that can satisfy a broad range of
laboratory requirements.
The QuantStudio™3D AnalysisSuite™Software is a web-based application that is
recommended for use with the following Internet browsers.
•Google™Chrome™v37 or later
•Microsoft™Internet Explorer™v10 or later
•Mozilla™Firefox™v32 or later
•Apple™Safari™v6 or later
Note: The software performance may vary based on your system conguration, and
requires an internet connection capable of 300kbps/300kbps (upload/download) or
better. If your network employs a rewall that restricts outbound trafc, it must be
congured to allow outbound access to apps.lifetechnologies.com on HTTPS-443.
Data collection
Supported
dyes
System
requirements
Chapter 1 Product information
About the software 1
QuantStudio™3D Digital PCR System User Guide 13

About the chip
The QuantStudio™3D Digital PCR 20K Chip v2 is a 10-mm2high-density reaction
plate that has a single array of 20,000 reaction wells. Hydrophobic coatings on the
chip surface enable loading and isolation of independent PCR reactions within the
wells.
Once loaded with a reaction, the chip must be sealed using the QuantStudio™3D
Digital PCR Chip Lid v2 and lled with Immersion Fluid. Figure 2 shows the
components of the chip assembly.
443MPJ
443MPJ
1
2
3
5
443MPJ
4
7
6
Figure 2 QuantStudio™3D Digital PCR 20K Chip v2
1QuantStudio™3D Digital PCR Chip Lid v2—The lid used to seal the chip for thermal cycling
and imaging.
2QuantStudio™3D Digital PCR 20K Chip v2—A 10-mm2chip with 20,000 wells that contain
the individual PCR reactions.
3Chip case—The thermal-conductive base that secures and protects the chip.
4Chip ID—A unique alphanumeric ID on the chip lid.
5Fill port—The hole in the chip case lid through which Immersion Fluid is injected onto the
chip.
6Reaction wells—The 20,000 physical holes on the surface of the chip that contain the
individual PCR reactions.
72D barcode—A barcode that encodes the same alphanumeric string as the Chip ID, scanned
by the QuantStudio™3D Digital PCR Instrument for chip tracking.
Chapter 1 Product information
About the chip
1
14 QuantStudio™3D Digital PCR System User Guide

Getting Started
For system installation instructions, see Appendix A, “Install the QuantStudio™3D
Digital PCR System”.
2
QuantStudio™3D Digital PCR System User Guide 15

Operational workflow
The following shows a single experiment workow on the QuantStudio™3D
Digital PCR System. The procedures for sample and/or consumable preparation and
result analysis can vary depending on the specic experiment that you are
performing.
Start
q
Set up the dPCR reaction by mixing sample, master mix, and
assay(s).
q
Load the dPCR reaction onto a QuantStudio™3D Digital PCR
20K Chip v2, apply the lid, load the assembly with Immersion
Fluid, then seal the loading port.
q
Perform the PCR using the ProFlex™2 × Flat PCR System or Dual
Flat Block GeneAmp™PCR System 9700.
q
Read the chip using the QuantStudio™3D Digital PCR Instrument.
q
Review the results on the instrument touchscreen.
q
Store or discard the chip.
q
Analyze the data using the QuantStudio™3D AnalysisSuite™
Software.
q
Finish
Note: See the user documentation for the QuantStudio™3D AnalysisSuite™Software
for detailed information on data analysis.
Chapter 2 Getting Started
Operational workflow
2
16 QuantStudio™3D Digital PCR System User Guide

Connect the QuantStudio™3D Digital PCR Instrument to a network
If you choose not to connect the QuantStudio™3D Instrument using the setup wizard
during installation, you can connect it to a network at any time afterwards to
streamline data transfer to the QuantStudio™3D AnalysisSuite™Software. See
Appendix D, “Networking” for the complete installation procedure, including
guidelines for integrating the instrument into your laboratory network.
IMPORTANT! This document does not provide adequate detail to integrate the
QuantStudio™3D Instrument into all possible network architectures. Because your
laboratory network can contain advanced features (such as rewalls), we recommend
that you consult a network administrator before connecting your instrument.
Connect the QuantStudio™3D AnalysisSuite™Software
You can connect to the QuantStudio™3D AnalysisSuite™Software before or after you
install the QuantStudio™3D Digital PCR Instrument. The software supports two
methods of deployment (server and Connect cloud-based platform), so the method
that you follow will depend upon the deployment you purchased. Regardless of the
deployment, you can use the AnalysisSuite™Software web application to analyze
data generated by your QuantStudio™3D Instrument. For more information, see the
user documentation for the QuantStudio™3D AnalysisSuite™Software.
Choosing a thermal cycler
IMPORTANT! Due to the unique physical characteristics of the QuantStudio™3D
Digital PCR 20K Chip v2, the ProFlex™2 × Flat PCR System and the GeneAmp™PCR
System 9700 are the only thermal cyclers approved to run prepared chips.
The QuantStudio™3D Digital PCR Instrument can image the QuantStudio™3D
Digital PCR 20K Chip v2 prepared using the ProFlex™2 × Flat PCR System or the
Dual Flat Block GeneAmp™PCR System 9700. Both automated thermal cyclers
perform nucleic acid amplication using the polymerase chain reaction (PCR) and are
designed to run the chip.
The ProFlex™2 × Flat PCR System interface consists of a touchscreen that is used
for both data entry and display. The GeneAmp™PCR System 9700 interface consists
of a control panel with a full numeric keypad, soft keys, and an LCD display screen
that shows run information. Both the ProFlex™PCR System and the GeneAmp™PCR
System 9700 use at sample block modules that install to the tops of the instrument
base modules. The sample block modules include two at aluminum sample blocks
that are designed to perform thermal cycling of up to 24 sealed chips simultaneously.
Both sample block modules include heated covers that are manually positioned over
the sample blocks before each PCR run. The covers apply pressure to the chips to
seat them rmly and precisely on the sample blocks, ensuring efcient heat transfer.
Compatible
thermal cyclers
Chapter 2 Getting Started
Connect the QuantStudio™3D Digital PCR Instrument to a network 2
QuantStudio™3D Digital PCR System User Guide 17

F1 F2 F3 F4
1 2 3
4 5 6
7 8 9
ENTER
STOP
0 CE
F5
GeneAmp®
PCR System 9700
POWER
ProFlex
PCRSystem
1
3
24
5
6
3
ProFlexTM Instrument GeneAmpTM Instrument
1USB port
2Touchscreen
3Heated cover
4LCD display
5Keypad
6Power button
Chapter 2 Getting Started
Choosing a thermal cycler
2
18 QuantStudio™3D Digital PCR System User Guide

Prepare the digital PCR reaction and
load the chips
■Guidelines for loading and sealing chips ................................ 19
■Prepare the DNA samples ............................................. 20
■Prepare the digital PCR reactions ...................................... 22
■Load the chips using the QuantStudio™3D Digital PCR Chip Loader ...... 24
Guidelines for loading and sealing chips
To ensure proper processing and analysis of loaded chips, handle them according to
the following guidelines:
•Always wear powder-free gloves when loading and sealing chips.
IMPORTANT! Never handle chips or chip lids without gloves. Oils from your
hands can contaminate the components and interfere with thermal cycling and
imaging.
•Hold the chip and lid gently by their sides.
–If you accidentally touch the chip surface, discard the chip.
–If you accidentally touch the chip lid window, clean it using a laboratory wipe
that has been sprayed with isopropanol, then dry by wiping in one direction
using a clean low-lint wipe.
443MPJ
•Use a permanent pen or marker to label the back of each loaded chip for sample
tracking.
•Load each chip within 2 hours after opening it.
•Load chips in alphanumeric order (according to the chip ID printed on the lid) to
avoid data entry errors.
3
QuantStudio™3D Digital PCR System User Guide 19

•Apply the chip lid and add Immersion Fluid immediately after loading each chip
to avoid evaporation.
•Load and seal chips in batches of up to 24 chips (the maximum number that can
be loaded onto one thermal cycler).
•If you do not intend to load an opened chip immediately, cover the chip using the
plastic plate included in the chip package to prevent contamination. If you plan
to keep the opened chip for longer than a day, the chip should be stored with
dessicant.
•Use all of the Immersion Fluid within 60 minutes of uncapping the syringe. After
a syringe is opened, you cannot recap it for later use.
•Thermal cycle the chips within 2 hours after loading them.
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
signicantly, sample preparation and template quality must be assessed on an
individual basis.
Use gDNA or cDNA template that:
•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 A260/230 and A260/280 ratio between 1.7 and 1.9
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
Quality of DNA
Quantity of
DNA
Chapter 3 Prepare the digital PCR reaction and load the chips
Prepare the DNA samples
3
20 QuantStudio™3D Digital PCR System User Guide