Applied Biosystems 3730xl User manual

For Research Use Only. Not for use in diagnostic procedures.
3730xl DNA Analyzer
USER GUIDE
3730xl Data Collection Software 5
Windows™10 Operating System
Catalog Number A39079
Publication Number 100077621
Revision A

Manufacturer: Life Technologies Holdings Pte Ltd | Block 33 | Marsiling Industrial Estate Road 3 | #07-06, Singapore 739256
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. 100077621
Revision Date Description
A 1 October 2018 New document.
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.
©2018 Thermo Fisher Scientific Inc. All rights reserved.

Contents
■CHAPTER 1 Instrument and software description ................... 10
Overview ...................................................................... 11
Parts of the instrument ......................................................... 12
Instrument interior components ................................................. 13
Instrument front panel indicators ................................................ 14
Consumables and usage limits .................................................. 16
Important notice regarding use of consumables that exceed supported limits ......... 16
Computer and software requirements ............................................ 17
Theory of operation ............................................................ 17
Preparing samples ......................................................... 17
Preparing the instrument ................................................... 18
During a run .............................................................. 18
Results ................................................................... 18
Overview of the software ........................................................ 19
Use the instrument with Thermo Fisher Connect ................................... 22
Secondary analysis software .................................................... 23
Data Collection software terminology ............................................. 24
Workflow for a typical run ....................................................... 26
■CHAPTER 2 Prepare the instrument .................................. 27
Power on the computer and the instrument ....................................... 27
Start the software .............................................................. 28
Prepare buffer and fill the reservoirs ............................................. 29
Required materials ........................................................ 29
1X run buffer storage ...................................................... 29
When to change the 1X run buffer ............................................ 30
Prepare the 1X run buffer ................................................... 30
Fill the water and buffer reservoirs .......................................... 30
Place reservoirs into the instrument .............................................. 32
Fill the anode buffer jar ......................................................... 33
Check the polymer level ........................................................ 34
Check for bubbles in the pump system ............................................ 35
Check the active spectral calibration ............................................. 36
3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide
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Prepare and load sample plates .................................................. 37
Default injection positions—48-capillary arrays .................................... 37
96-Well plate, 48 capillaries ................................................. 37
384-Well plate, 48 capillaries ............................................... 38
Default injection positions—96-capillary arrays .................................... 39
96-Well plate, 96 capillaries ................................................. 39
384-Well plate, 96 capillaries ............................................... 40
Seal and assemble plates ....................................................... 41
Seal and prepare the plate assemblies ....................................... 41
Prepare the plate assemblies ............................................... 42
Important heat seal recommendations ....................................... 44
Place plate assemblies into the instrument ........................................ 44
■CHAPTER 3 Use the instrument with Thermo Fisher Connect ..... 46
Thermo Fisher Connect features ................................................. 46
Register and obtain a Thermo Fisher Connect account .............................. 47
Connect the instrument to your Thermo Fisher Connect account ..................... 47
Set up the data storage location and email notifications ............................. 48
Monitor a run from InstrumentConnect ........................................... 49
Monitor a run from a mobile device ............................................... 50
View notifications from the instrument on the Cloud ................................ 51
For more information on using InstrumentConnect ................................. 52
Thermo Fisher Connect administrators for an instrument ........................... 52
First user who links is assigned administrator role ............................ 52
Instrument administrator functions .......................................... 52
Manage the users and administrators of your instrument ....................... 53
Disconnect individual users from an instrument ............................... 54
■CHAPTER 4 Create results groups and plate records using
default settings ........................................................... 55
Create a results group (using defaults) ........................................... 55
Create a plate record ........................................................... 56
■CHAPTER 5 Run the instrument ....................................... 58
Understanding run modes and default injection positions ........................... 59
Manual and auto run mode features .......................................... 59
Default injection order on plates ............................................. 59
Run the instrument in auto mode ................................................ 61
Run the instrument in manual mode .............................................. 61
Change injection order of plate quadrants ......................................... 63
Move a plate in the Input stack during a run ....................................... 64
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

Controlling the run ............................................................. 64
Monitor the status of the run .................................................... 65
Add reinjections to a plate record ................................................ 66
Check instrument status ........................................................ 67
Events box ................................................................ 67
Errors box ................................................................ 67
View the Event Log ............................................................. 68
View data from the run .......................................................... 69
View electropherogram data ................................................ 69
View electrophoresis conditions (EPT) ........................................ 71
View data from a completed run (Run History)................................. 71
Check the autoextraction status and manually re-extract ........................... 72
Autoextraction and re-extraction ............................................ 72
Re-extract runs ........................................................... 72
■CHAPTER 6 Create and manage plate records and
results groups ............................................................ 74
Overview of plate records ....................................................... 74
Create and manage plate records ................................................ 76
Search for a plate record ........................................................ 77
Import a plate record ........................................................... 78
Overview of results groups ...................................................... 79
Create and manage results group ................................................ 80
Create a results group (detailed procedure) ....................................... 80
Specify custom sample file naming in a results group .............................. 82
■CHAPTER 7 Create and manage instrument protocols, run
modules, and analysis protocols ........................................ 84
Create and manage instrument protocols ......................................... 84
Create an instrument protocol ................................................... 85
Create run module ............................................................. 86
Run module parameters .................................................... 87
Overview of analysis protocols (sequence analysis) ................................. 87
Create and manage analysis protocols ............................................ 88
Create an analysis protocol ...................................................... 89
Analysis protocol parameters ............................................... 89
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■CHAPTER 8 Calibrate the instrument ................................. 91
Perform spatial calibration ...................................................... 91
Spatial calibration description and run time ................................... 91
When to perform a spatial calibration ........................................ 91
Perform spatial calibration ................................................. 91
Evaluate the spatial calibration data ......................................... 92
Examples of passing and failing spatial profiles ................................ 94
Magnify the spatial profile .................................................. 96
Perform spectral calibration for sequencing and fragment analysis .................. 97
Spectral calibration definition and run time ................................... 97
When to perform the calibration ............................................. 97
Supported dye sets and calibration standards ................................. 98
Required materials ........................................................ 98
Prepare the spectral calibration standard ..................................... 99
Create a spectral instrument protocol ........................................ 99
Create a spectral calibration plate .......................................... 100
Run the spectral calibration plate ........................................... 101
Place plate assemblies into the instrument .................................. 102
View the Pass/Fail status after the run ...................................... 103
Evaluate the spectral calibration data ....................................... 105
Magnify the spectral profile ................................................ 108
Examples of passing sequencing spectral calibrations ........................ 110
Example of a passing fragment analysis spectral calibration ................... 111
Select a previous calibration as the active spectral calibration .................. 111
■CHAPTER 9 Maintenance ............................................. 113
Maintenance schedules ........................................................ 113
Daily maintenance ........................................................ 113
Weekly maintenance ...................................................... 114
Monthly maintenance ..................................................... 115
As-Needed maintenance .................................................. 115
Wizards types and functions .................................................... 116
Typical conditions for using maintenance wizards ................................. 117
Effects of the instrument door state on wizard tasks .......................... 118
Manual control of autosampler content ...................................... 118
Clean the instrument .......................................................... 119
Replenish or change polymer type .............................................. 119
Before using the polymer .................................................. 119
Replenish the polymer .................................................... 119
Change polymer type ...................................................... 120
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

Clean the polymer delivery pump (PDP) and lower polymer block ................... 121
Parts of the polymer delivery pump ......................................... 121
Guidelines ............................................................... 121
Frequency ............................................................... 121
Clean the polymer delivery pump chamber, channels, and tubing ............... 122
Flush the water seal trap ...................................................... 122
Install the capillary array ...................................................... 123
Required materials ....................................................... 123
Install a new or used capillary array ........................................ 123
Store a capillary array ......................................................... 124
Clean the detection cell ........................................................ 125
Perform a short-term shutdown ................................................ 126
Materials required ........................................................ 126
Perform a short-term shutdown ............................................ 127
Maintain adequate space for database and sample data storage .................... 130
Pre-run automatic disk space checks ....................................... 130
Full database error ....................................................... 131
Check available disk space on E: drive ....................................... 131
Delete data from the database .............................................. 132
■APPENDIX A Troubleshooting ........................................ 134
Restart the instrument and the computer ........................................ 134
Display the Event Log ......................................................... 135
Review a run log file .......................................................... 135
Hi‑Di™Formamide preparation and storage ...................................... 136
Front panel status light troubleshooting ......................................... 136
Instrument run troubleshooting ................................................. 137
Re-extraction troubleshooting .................................................. 137
Service console troubleshooting ................................................ 138
Spatial calibration troubleshooting .............................................. 138
Spectral calibration troubleshooting ............................................. 139
General troubleshooting ....................................................... 140
■APPENDIX B Instrument specifications ............................. 142
Instrument specifications ...................................................... 142
Environmental requirements ................................................... 143
Electrical requirements ........................................................ 144
Power and communication connections .......................................... 145
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■APPENDIX C Run modules ........................................... 146
Fragment analysis run times ................................................... 146
Sequence analysis run times ................................................... 146
■APPENDIX D Dye sets: G5, G5-RCT, Any4Dye, Any4Dye-HDR,
and Any5Dye ............................................................. 148
Supported dye sets and calibration standards .................................... 148
Dye sets G5 and G5-RCT for fragment analysis .................................... 149
Fragment analysis using a 96-capillary array: cross-talk peaks ................. 149
Reduced cross-talk—Dyeset G5-RCT ........................................ 149
When to use RCT dye sets .................................................. 149
■APPENDIX E Catalog numbers ....................................... 150
Plates, bases, retainers, and septa .............................................. 150
Instrument consumables ...................................................... 151
Sequencing and fragment analysis calibration standards ........................... 151
Fragment analysis size standards ............................................... 152
■APPENDIX F Safety .................................................... 153
Symbols on this instrument .................................................... 153
Safety symbols ........................................................... 153
Location of safety labels ................................................... 155
Control and connection symbols ............................................ 156
Conformity symbols ....................................................... 156
Safety information for instruments not manufactured by Thermo Fisher Scientific .... 157
Instrument safety ............................................................. 157
General ................................................................. 157
Physical injury ........................................................... 157
Electrical safety .......................................................... 158
Cleaning and decontamination ............................................. 159
Instrument component and accessory disposal ............................... 159
Laser safety .............................................................. 160
Safety and electromagnetic compatibility (EMC) standards ......................... 160
Safety standards .......................................................... 161
EMC standards ........................................................... 161
Environmental design standards ............................................ 162
Chemical safety ............................................................... 163
Biological hazard safety ........................................................ 164
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

■Documentation and support ........................................... 165
Related documentation ........................................................ 165
Customer and technical support ................................................ 165
Limited product warranty ...................................................... 166
Index ..................................................................... 167
Contents
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Instrument and software description
■Overview ........................................................... 11
■Parts of the instrument ................................................ 12
■Instrument interior components ........................................ 13
■Instrument front panel indicators ....................................... 14
■Consumables and usage limits ......................................... 16
■Important notice regarding use of consumables that exceed supported
limits ............................................................... 16
■Computer and software requirements ................................... 17
■Theory of operation .................................................. 17
■Overview of the software .............................................. 19
■Use the instrument with Thermo Fisher Connect ......................... 22
■Secondary analysis software ........................................... 23
■Data Collection software terminology ................................... 24
■Workow for a typical run ............................................. 26
1
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

Overview
The Applied Biosystems™ 3730xl DNA Analyzer with 3730xl Data Collection Software
5 is a uorescence-based DNA analysis instrument. It uses capillary electrophoresis
technology with 48 or 96 capillaries.
The instrument is designed for high-throughput, unaended operation. Automation
features include an integrated plate stacker and internal bar code reader.
The analyzer (Cat. No. A41046) is shipped with the following components.
• 48-capillary or 96-capillary array and POP™ polymer
• Reagents and consumables for your application and for system qualication
• Computer workstation and monitor
• Integrated software for instrument control and data collection.
• Optional secondary analysis software. See “Secondary analysis software“ on
page 23.
IMPORTANT! The protection provided by the equipment may be impaired if any of
the following conditions occur.
·The instrument is operated outside the environment and use specications
·The user provides inadequate maintenance
·The equipment is used in a manner not specied by the manufacturer (Thermo
Fisher Scientic)
IMPORTANT! Observe current good laboratory practices when using this instrument.
Chapter 1 Instrument and software description
Overview
1
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Parts of the instrument
5
3
4
1 2
7
6
Figure 1 3730xl DNA Analyzer
1Polymer delivery pump (PDP). See Figure 3.
2Oven door (The detector cell and capillary array are inside this compartment. )
3Stacker (automated plate handler)
4Stacker door indicator light
5Power button and status lights
6Tray button
7Buffer, water, and waste reservoirs
Chapter 1 Instrument and software description
Parts of the instrument
1
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

Instrument interior components
1 2 3
Figure 2 Polymer delivery pump, detection cell, and capillary array (view with oven door
open)
1Polymer delivery pump
2Detection cell (behind door)
3Capillary array
1
2
3
6
7
4
5
Figure 3 Polymer delivery pump (PDP)
1Pump block
2Polymer supply tube
3Polymer reservoir
4Anode buffer jar
5Capillary array
6Interconnect tube
7Lower polymer block
Chapter 1 Instrument and software description
Instrument interior components
1
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Instrument front panel indicators
1
2
1Status indicators
2Stacker indicators
Table 1 Status lights
Status Light Status
Solid Green • The instrument is ready
•An automated wizard operation is in progress with the
instrument door closed
Flashing Green • A run is in progress
Solid Yellow • The instrument cannot communicate with the computer. See
Appendix A, “Troubleshooting“.
• The buffer reservoir or capillary array is not installed
Flashing Yellow • The instrument is performing diagnostics
•The instrument door or oven door is open
• An automated wizard operation is in progress with the
instrument door open
• The instrument is downloading firmware
Solid Red • The instrument has detected a problem. See Appendix A,
“Troubleshooting“.
• Restart the instrument and computer. See “Power on the
computer and the instrument“ on page 27.
Chapter 1 Instrument and software description
Instrument front panel indicators
1
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

Table 2 Stacker indicators
Status Light Status
Solid Green •In Stack— At least one plate with an unknown barcode
number is in the In stack, and
•Out Stack— Empty, no plates in stack
Solid Red •In Stack—Empty, no plates in stack
and
•Out Stack— Full, remove plates before you can start
another run
Chapter 1 Instrument and software description
Instrument front panel indicators
1
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Consumables and usage limits
Consumable Storage On-instrument limit
Polymer 2–8°C 7 days
1X run buffer (prepared from
3730 Running Buffer (10X) )
Used in the anode buffer jar and
the buffer reservoir
• Shipped at room
temperature. Stable for
1 week.
• 2–8°C for up to 1 month
48 hours
IMPORTANT! Do not add buffer to the anode
buffer jar or the buffer reservoir. Always replace
with fresh buffer.
Capillary array Room temperature 300 injections
Important notice regarding use of consumables that exceed
supported limits
Life Technologies does not recommend the use of consumables that exceed supported
limits. The recommended limits are designed to promote the production of high
quality data and minimize instrument downtime. Reagent and consumable lifetime
minimum performance are based on testing and studies that use reagents and
consumables that have not exceeded supported limits.
The use of consumables beyond the supported limits may impact data quality or
cause damage to the instrument or capillary array. The cost of repairing such damage
is NOT covered by any Life Technologies product warranty or service plan. Customer
use of expired consumables is at customer's own risk and without recourse to Life
Technologies. For example, product warranties do not apply to defects resulting from
or repairs required due to misuse, neglect, or accident including, without limitation,
operation outside of the environmental or use specications or not in conformance
with Life Technologies instructions for the instrument system, software, or
accessories.
Please see your specic service contract or limited product warranty for exact
language regarding coverage and ask your Life Technologies representative if you
have further questions.
Chapter 1 Instrument and software description
Consumables and usage limits
1
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

Computer and software requirements
Computer/software Requirement
Computer IMPORTANT! Do not modify the instrument hardware or software without notifying
Thermo Fisher Scientific. Any modifications must be made by Thermo Fisher Scientific
under change control.
The computer provided with the instrument contains validated software and settings.
Do not change the static IP settings.
For minimum computer requirements, see “Instrument specifications“ on page 142.
IMPORTANT! Do not rename the computer after the 3730xl Data Collection Software
5 is installed. The instrument computer has been assigned a unique name. Changing
the name may cause the software to malfunction.
Because of the Ethernet cable connection between the instrument and the computer,
the computer must be located within 9 feet of the instrument.
IMPORTANT! The instrument communicates with the computer by Ethernet
connection. Do not make any changes to the computer ethernet/internet connections
during a run or during calibration.
Operating system Operating system: Windows™10 Enterprise 2016 LTSB, 64-bit (requires 5 partitions)
Consult with a Thermo Fisher Scientific representative before updating the Windows™
operating system or firewall settings.
Instrument firmware Instrument firmware is to be updated only by a Thermo Fisher Scientific representative.
Antivirus software
requirements
No antivirus software is provided with the Data Collection software. The following
applications are compatible with the software:
• Symantec Endpoint Protection 12
• McAfee Endpoint Security version 10.5
• Windows Defender Antivirus (comes as part of the Windows™10 installation)
Other software CAUTION! Do not install additional software on the computer other than
antivirus software. Changes to the configured software could void the
instrument warranty and cause the instrument software to be non-operational.
Theory of operation
When DNA samples are prepared for sequencing and fragment analysis on the
instrument, uorescent dyes are aached to the DNA.
Preparing
samples
Chapter 1 Instrument and software description
Computer and software requirements
1
3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide
17

Two calibrations are required to prepare the instrument for sample runs:
•Spatial calibration—Determines the position of the image from each capillary on
the CCD array. For more information, see “Perform spatial calibration“ on
page 91.
•Spectral calibration—Generates a matrix for each capillary that compensates for
dye overlap and is used to convert the 20-color data into 4-, 5-, or 6-dye data. For
more information, see “Perform spectral calibration for sequencing and fragment
analysis“ on page 97.
During a run, the instrument performs the following steps.
• Prepares the capillaries by pumping fresh polymer under high pressure from the
polymer delivery pump to the waste reservoir.
• Electrokinetically injects the sample into the capillaries by briey applying low
voltage.
• Washes the capillary tips in the water reservoir, then returns the capillary to the
buer reservoir.
• Ramps the voltage up to a constant level.
A high electric eld is created between the ground end of the electrode in the
anode buer jar and the negative voltage that is applied to the load header of the
capillary array. This eld pulls the negatively charged DNA through the
separation polymer. The smaller fragments migrate faster than the larger
fragments and reach the detector rst.
To ensure optimal separation and maintain denaturation of the DNA, the
capillaries are thermally controlled in the oven and in the detection cell.
In the detection cell, the dyes that are aached to DNA are excited by a narrow
beam of laser light. The laser light is directed into the plane of the capillaries from
both the boom and top. A small amount of laser light is absorbed by the dyes
and emied as longer wavelength light in all directions.
• CCD captures the uorescent light on the instrument optics while blocking the
laser light. The light passes through a transmission grating, which spreads out
the light. The light is imaged onto a cooled CCD array.
• Computer converts the data into multi-dye data for the entire run. For
sequencing applications, 4 dierent dyes are used to determine the 4 bases A, G,
C, and T. For fragment analysis applications, up to 6 dyes can be used in a single
run for higher throughput.
• Stores the data in an internal database, then creates sequence analysis (AB1) and
fragment analysis (FSA) data les by "autoextracting" data from the database.
The software generates an electropherogram (intensity plot) for each dye based on the
migration of DNA fragments over the run and generates primary analysis results:
• For sequencing applications, the electropherogram is adjusted to compensate for
slight mobility dierences due to the dyes, then basecalling is performed and
quality values are assigned.
• For fragment analysis, the software uses the internal size standard to assign a
fragment size and a sizing quality value to each peak.
Preparing the
instrument
During a run
Results
Chapter 1 Instrument and software description
Theory of operation
1
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide

Overview of the software
3730xl Data Collection Software 5 manages instrument setup, controls instrument
operations, allows real-time data visualization, and performs diagnostics. The Thermo
Fisher Connect feature allows you link the instrument to your Thermo Fisher Connect
account and upload data.
Use the Navigation pane (Figure 4) to set up your run, view and interrogate results,
or to determine the amount of free space remaining in your database.
Figure 4 Navigation pane
Chapter 1 Instrument and software description
Overview of the software
1
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19

1
2
3
4
5
6
7
8
Figure 5 Navigation pane expanded
1Results Group—Determine the naming and storage location of results. See “Create a
results group (using defaults)“ on page 55.
2Database Manager—Manage the database; view number of runs in the database and
available disk space. See “Maintain adequate space for database and sample data
storage“ on page 130.
3Thermo Fisher Connect—Link to your Thermo Fisher Connect account to set up, view, and
analyze data. See Chapter 3, “Use the instrument with Thermo Fisher Connect“.
4Plate Manager—Create and manage plate records. See Chapter 4, “Create results groups
and plate records using default settings“ and “Create and manage plate records“ on
page 76.
5Protocol Manager—Create and manage instrument protocols, which specify run modules,
dye sets, capillary length, polymer, and chemistry type. See Chapter 7, “Create and
manage instrument protocols, run modules, and analysis protocols“.
6Module Manager— Create and manage run modules, which specify instrument settings.
See Chapter 7, “Create and manage instrument protocols, run modules, and analysis
protocols“.
7Run History—View previous runs. See “View data from a completed run (Run History)“ on
page 71.
8
Instrument name —View the status of the instrument. See “Check instrument status“ on
page 67.
Chapter 1 Instrument and software description
Overview of the software
1
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3730xl DNA Analyzer with 3730xl Data Collection Software 5 User Guide