Synergy™ 2

Synergy™ 2
Multi-Detection Microplate Reader
Synergy™ 2
Operator’s Manual
Synergy™ 2
Multi-Mode Microplate Reader
Operator’s Manual
© 2010
Part Number 7131000
Revision E
BioTek® Instruments, Inc.
2 | Preface
Notices
BioTek® Instruments, Inc.
Highland Park, P.O. Box 998
Winooski, Vermont 05404-0998 USA
All Rights Reserved
© 2010, BioTek® Instruments, Incorporated. No part of this publication may be
reproduced, transcribed, or transmitted in any form, or by any means electronic or
mechanical, including photocopying and recording, for any purpose other than the
purchaser’s use without written permission of BioTek Instruments, Inc.
Trademarks
BioTek® is a registered trademark, and Synergy™ 2, Gen5™, Take3™, and BioStack™
are trademarks of BioTek Instruments, Inc. Glowell™ is a trademark of LUX
Biotechnology, Ltd. Harta™ is a trademark of Harta Instruments. FluoSpheres® is a
registered trademark of Invitrogen Corporation.
Microsoft®, Windows®, Windows XP, Windows 2000, and Windows Vista™ are either
registered trademarks or trademarks of Microsoft Corporation in the United States
and/or other countries.
All other trademarks are the property of their respective holders.
Restrictions and Liabilities
Information in this document is subject to change and does not represent a
commitment by BioTek Instruments, Inc. Changes made to the information in this
document will be incorporated in new editions of the publication. No responsibility is
assumed by BioTek for the use or reliability of software or equipment that is not
supplied by BioTek or its affiliated dealers.
BioTek Instruments, Inc.
Notices |
Contents
Notices ............................................................................................... 2
All Rights Reserved.......................................................................... 2
Trademarks.................................................................................... 2
Restrictions and Liabilities ................................................................ 2
Contacting BioTek Instruments, Inc. ....................................................... 8
Customer Service and Sales ............................................................. 8
Service/TAC ................................................................................... 8
European Coordination Center/Authorized European Representative ....... 8
Revision History ................................................................................... 9
Document Conventions ........................................................................11
Intended Use Statement ......................................................................11
Quality Control ...................................................................................11
Warranty and Product Registration ........................................................12
Warnings ...........................................................................................12
Hazards .............................................................................................12
Precautions ........................................................................................13
CE Mark.............................................................................................14
Directive 89/336/EC: Electromagnetic Compatibility ............................14
Directive 73/23/EEC Low Voltage (Safety) .........................................15
Directive 2002/96/EC: Waste Electrical and Electronic Equipment .........15
Directive 98/79/EC: In Vitro Diagnostics............................................15
Electromagnetic Interference and Susceptibility .......................................16
User Safety ........................................................................................16
Safety Symbols...................................................................................17
Chapter1: Introduction ..........................................................................19
Product Description .............................................................................20
Package Contents................................................................................21
Optional Accessories ............................................................................22
Product Support and Service.................................................................24
Technical Assistance Center (TAC) ....................................................24
Applications Support.......................................................................24
Chapter 2: Installation ...........................................................................25
Product Registration ............................................................................26
Important Information .........................................................................26
1: Unpack and Inspect the Reader .........................................................27
2: Unpack and Inspect the Dispense Module ...........................................28
3: Select an Appropriate Location ..........................................................29
4: Remove the Shipping Hardware ........................................................29
5: Install the Power Supply...................................................................34
6: Install the Dispense Module ..............................................................35
7: Connect the Host Computer ..............................................................37
8: Install Gen5 on the Host Computer ....................................................38
Synergy 2 Operator’s Manual
3
4 | Preface
9: Turn on the Power Supply and Reader................................................38
10: Establish Communication ................................................................39
Communication Errors ....................................................................39
11: Run a System Test ........................................................................39
12: Test the Injector System ................................................................41
Operational/Performance Qualification....................................................42
Repackaging and Shipping Instructions ..................................................43
Chapter 3: Getting Started .....................................................................49
Modular Design...................................................................................50
External Components...........................................................................51
Internal Components ...........................................................................52
Tungsten Lamp ..............................................................................53
Description and Location ............................................................53
When to Replace the Lamp .........................................................53
Excitation/Emission Filters ...............................................................54
EX/EM Configuration for Luminescence .........................................54
Mirrors .........................................................................................55
Injector System ..................................................................................56
External Dispense Module................................................................56
Internal Tubing ..............................................................................57
Priming the Injector System ............................................................58
Gen5 Software....................................................................................59
Define Excitation/Emission Filters .....................................................59
Define Mirrors................................................................................59
Protocols and Experiments...............................................................60
Dispense Module Control .................................................................61
Prime ......................................................................................61
Purge ......................................................................................61
Recommendations for Optimum Performance ..........................................62
Chapter 4: Filters and Mirrors ................................................................63
Excitation/Emission Filters ....................................................................64
Change the Filter Wheels and Filters .................................................65
Clean the Filters.............................................................................66
Filters Available from BioTek ............................................................67
Mirrors ..............................................................................................68
Change the Mirror Holder and Mirrors................................................71
To remove the mirror holder .......................................................72
To change a mirror in the mirror holder ........................................73
To reinstall the mirror holder ......................................................76
Clean the Mirrors ...........................................................................76
Mirrors Available from BioTek...........................................................77
Chapter 5: Instrument Qualification.......................................................79
Overview ...........................................................................................80
IQ/OQ/PQ ..........................................................................................80
Recommended Qualification Schedule ....................................................82
BioTek Instruments, Inc.
Notices |
System Test .......................................................................................83
Absorbance Plate Test..........................................................................87
Description....................................................................................87
Test Plate Certificates .....................................................................88
Define Absorbance Test Plate Parameters ..........................................88
Run the Absorbance Plate Test .........................................................89
Sample Report...............................................................................90
Results & Troubleshooting Tips.........................................................91
Absorbance Liquid Tests.......................................................................92
Absorbance Liquid Test 1.................................................................93
Materials ..................................................................................93
Solution A ...........................................................................93
Solution B ...........................................................................93
Prepare the Plate ......................................................................94
Read the Plate ..........................................................................94
Analyze the Results ...................................................................94
Absorbance Liquid Test 2.................................................................95
Materials ..................................................................................95
Prepare the Dilutions .................................................................95
Prepare the Plate ......................................................................96
Linearity & Repeatability Tests ....................................................96
Alignment Test .........................................................................96
Absorbance Liquid Test 3 (Optional)..................................................97
Materials ..................................................................................97
Buffer Solution.....................................................................97
Prepare the Plate ......................................................................98
Read the Plate ..........................................................................98
Analyze the Results ...................................................................98
Fluorescence Liquid Tests .....................................................................99
Required Materials........................................................................ 100
All Tests................................................................................. 100
Corners/Sensitivity/Linearity (FI) Tests ...................................... 100
Fluorescence Polarization (FP) Test ............................................ 101
Time-Resolved Fluorescence (TRF) Test ...................................... 101
Test Solutions.............................................................................. 101
Corners/Sensitivity/Linearity (FI) Tests ...................................... 102
Fluorescence Polarization (FP) Test ............................................ 103
Time-Resolved Fluorescence (TRF) Test ...................................... 103
Procedure ................................................................................... 104
Pipette Maps................................................................................ 105
Corners Test........................................................................... 105
Sensitivity and Linearity Tests ................................................... 106
FP Test .................................................................................. 107
TRF Test ................................................................................ 107
Results Analysis ........................................................................... 108
Synergy 2 Operator’s Manual
5
6 | Preface
Corners Test........................................................................... 108
Sensitivity Test ....................................................................... 108
Linearity Test.......................................................................... 108
Fluorescence Polarization (FP) Test ............................................ 109
Time-Resolved Fluorescence (TRF) Test ...................................... 109
Troubleshooting Tips..................................................................... 110
Gen5 Protocol Reading Parameters ................................................. 111
Fluorescence Tests Using Methylumbelliferone.................................. 114
Required Materials................................................................... 114
Test Solutions......................................................................... 114
Procedure .............................................................................. 115
Pipette Map ............................................................................ 116
Results Analysis ...................................................................... 117
Sensitivity Test .................................................................. 117
Linearity Test..................................................................... 117
Gen5 Protocol Reading Parameters ............................................ 118
Luminescence Test ............................................................................ 119
Harta Plate Test ........................................................................... 119
Materials ................................................................................ 119
Procedure .............................................................................. 119
Plate Map ............................................................................... 119
Results Analysis ...................................................................... 120
Glowell Test ................................................................................ 120
Materials ................................................................................ 120
Procedure .............................................................................. 120
Results Analysis ...................................................................... 121
Troubleshooting ........................................................................... 122
Gen5 Protocol Reading Parameters ................................................. 122
Dispense Module Tests....................................................................... 124
Required Materials........................................................................ 124
Test Solutions.............................................................................. 125
Procedure for Models with the Absorbance Module ............................ 126
Procedure for Models without the Absorbance Module ........................ 127
Results Analysis ........................................................................... 128
Failures.................................................................................. 129
Gen5 Test Protocols for Models with the Absorbance Module............... 129
Gen5 Test Protocols for Models without the Absorbance Module .......... 131
Create the Dispense Protocols ................................................... 131
Create the Read Protocol (if needed).......................................... 132
Chapter 6: Preventive Maintenance......................................................137
Preventive Maintenance ..................................................................... 138
Daily Cleaning for the Dispense Module ........................................... 138
Schedule..................................................................................... 139
Warnings and Precautions .................................................................. 139
Clean Exposed Surfaces ..................................................................... 141
BioTek Instruments, Inc.
Notices |
Inspect/Clean Excitation and Emission Filters ........................................ 141
Inspect/Clean Mirrors ........................................................................ 142
Materials..................................................................................... 143
Procedure ................................................................................... 143
Flush/Purge the Fluid Path.................................................................. 143
Run a Dispense Protocol (Optional) ...................................................... 144
Empty/Clean the Tip Priming Trough .................................................... 145
Clean the Priming Plate ...................................................................... 145
Clean the Internal Components ........................................................... 146
Required Materials........................................................................ 146
Remove the Reader’s Shroud ......................................................... 147
Clean the shroud’s air filters ..................................................... 148
Remove the Internal Dispense Tubes and Injector Heads ................... 149
Clean the Dispense Tubes and Injector Heads .................................. 152
Clean Inside the Reader ................................................................ 153
Remove the Incubator Housing ...................................................... 154
Clean the Reader’s Surface ............................................................ 157
Reassemble the Components ......................................................... 158
Verify Performance ....................................................................... 159
Chapter 7: As Needed Maintenance ......................................................161
Decontamination............................................................................... 162
Required Materials........................................................................ 163
Procedure for Models without Injectors................................................. 164
Procedure for Models with Injectors ..................................................... 165
Routine Procedure ........................................................................ 165
Clean Exposed Surfaces ........................................................... 165
Decontaminate the Fluid Lines................................................... 166
Rinse the Fluid Lines ................................................................ 166
Clean the Internal Tubing and Injectors ...................................... 167
Decontaminate the Tip Priming Trough and Priming Plate .............. 167
Alternate Procedure ...................................................................... 167
Replace the Tungsten Lamp ................................................................ 169
Replace a Syringe ............................................................................. 171
Appendix A: Specifications ...................................................................173
General Specifications........................................................................ 174
Absorbance Specifications .................................................................. 175
Luminescence Specifications ............................................................... 176
Fluorescence Specifications................................................................. 177
Models with Injectors......................................................................... 179
Appendix B: Error Codes.......................................................................181
Overview ......................................................................................... 182
Contact Info: BioTek Service/TAC ................................................... 182
Error Codes ...................................................................................... 183
Appendix C: Instrument Dimensions for Robotic Interface ..................187
Synergy 2 Operator’s Manual
7
8 | Preface
Contacting BioTek Instruments, Inc.
BioTek® Instruments, Inc.
Highland Park, P.O. Box 998
Winooski, Vermont 05404-0998 USA
Customer Service and Sales
Internet:
www.biotek.com
Phone:
888-451-5171 (toll free in the U.S.)
802-655-4740 (outside the U.S.)
Fax:
802-655-7941
E-Mail:
customercare@biotek.com
Service/TAC
Phone:
800-242-4685 (toll free in the U.S.)
802-655-4740 (outside the U.S.)
Fax:
802-654-0638
E-Mail:
tac@biotek.com
European Coordination Center/Authorized European
Representative
BioTek® Instruments GmbH
Kocherwaldstrasse 34
D-74177 Bad Friedrichshall
Germany
Internet:
www.biotek.de
Phone:
+49 (0) 7136 9680
Fax:
+49 (0) 7136 968 111
E-Mail:
info@biotek.de
BioTek Instruments, Inc.
Revision History |
9
Revision History
Revision
Date
Changes
A
08/2006
First issue.
B
12/2007
General: Changed all instances of “multi-detection” to “multi-mode”.
Changed all instances of “filter cartridge” to “filter wheel”.
Preface: Added Glowell™ and FluoSpheres® to Trademarks. Removed
Figures list.
Ch. 1, Introduction: Updated Package Contents and Accessories lists.
Added note stating that part numbers are subject to change.
Ch. 2, Installation: Revised unpacking/repackaging instructions.
Added note stating that packaging materials are subject to change.
Ch. 3, Getting Started: Simplified Gen5 Software usage instructions.
Ch. 4, Filters and Mirrors: Mirrors section: Added information on
Visible and UV-polarizing filters. Corrected high end of EM Range for
510 nm mirror (changed 780 nm to 640 nm). Changed mirror label
location from “lower left corner” to “face up and readable” and added
an illustration. Updated list of dichroic mirrors available from BioTek.
Ch. 5, Instrument Qualification: Added materials and procedure for a
Luminescence Test. Fluorescence Liquid Tests section: Added option
to use Greiner SensoPlate to FI tests for the top optics. Corrected
instances of “505 nm” mirror to “510 nm” and “410 nm” to “400 nm”.
Under “Results Analysis” for the FP Test, corrected the wells used for
the Mean Blank calculations—changed from A1-H6 to A6-H6. Added
option to use Sodium Borate instead of PBS. Reconfigured the SF test
solutions and dilutions for efficiency and consistency with other
BioTek products. Added troubleshooting tip to reduce the Sensitivity
value in the Gen5 protocol if well(s) are overranging during the
Fluorescence Intensity tests. To minimize the opportunity for
overranging during these tests, reduced the recommended Sensitivity
values for the SF Fluorescence Intensity Corners and Sensitivity top
probe tests to 75. Updated the materials list and solution preparation
steps for the TRF test to support the use of FluoSpheres® from
Invitrogen. Removed screen shots of sample BioTek data sheets.
Under “Gen5 Protocol Reading Parameters” added the Sensitivity
value as an example of a protocol parameter that may need to be
adjusted for some readers. Added FI tests using Methylumbelliferone.
Ch. 6, Preventive Maintenance: Removed unnecessary task to clean
supply bottles.
Ch. 7, As Needed Maintenance: Changed the replacement tungsten
lamp part number to 7080500.
Appendix B, Error Codes: Revised to focus on only the most common,
most easily-fixed error codes that may appear in Gen5.
Synergy 2 Operator’s Manual
10 | Preface
Revision
Date
Changes
C
1/2010
Throughout: Added information for purchasing and using the Harta
Luminometer Reference Microplate. Added support for the BioTek
Take3 Multi-Volume Plate.
Preface: Updated the Intended Use Statement; the Synergy 2 may be
used for In Vitro Diagnostic, research and development, or other nonclinical purposes. Added information for registering products online
through the BioTek Customer Resource Center. Updated the list of
Hazards and Precautions. Updated CE Mark information and Safety
Symbols.
Ch 1, Introduction: Updated the list of Optional Accessories and
added a reference to the BioTek online Accessories search tool.
Removed the BioTek shipping address; customers will be notified of
the address when they contact TAC/Service for a Return Materials
Authorization number.
Ch 2, Installation: Combined reader unpacking/inspection and
shipping panel removal instructions. Moved dispense module figures
from step 2 to “Repackaging and Shipping Instructions.” Corrected
USB Driver Software CD instructions under “Establish
Communication”.
Ch 3, Getting Started: Added “Modular Design,” “External
Components,” and “Internal Components.” Clarified/updated content
in remaining sections.
Ch 4, Filters and Mirrors: Clarified instructions throughout. Added
“Filters Available From BioTek.” Moved mirror cleaning instructions to
Chapter 7.
Ch 5, Instrument Qualification: Corrected unit of measure typos.
Added reference to new BioTek test kits for fluorescence liquid
testing. Added Luminescence Test using the new Harta plate.
Corrected Accuracy % Error calculation for Dispense Module tests.
Ch 6, Preventive Maintenance: Added instructions for inspecting/
cleaning mirrors.
Appendix A, Specifications: Added support for the Take3 plate.
Appendix B, Error Codes: Updated error code descriptions.
D
3/2010
Preface: Updated Directive 98/79/EC: In Vitro Diagnostics.
Ch 2, Installation: Corrected typo in the step heading numbers.
Ch 5, Instrument Qualification: For the luminescence test using the
Harta Luminometer Reference Microplate, moved the background
(buffer) wells from rows C and D to rows F and G. For the background
read step, changed the Top Probe Vertical Offset to 4.00 mm.
Ch 6, Preventive Maintenance: Under “Run a Dispense Protocol
(Optional),” added instruction to set the Plate Type in the Gen5
protocol to match the plate being used for the test.
E
4/2010
Ch 5, Instrument Qualification: For the luminescence test using the
Harta Luminometer Reference Microplate, corrected errors in the
Gen5 protocol reading parameter table on page 123 (read step 3).
BioTek Instruments, Inc.
Document Conventions |
11
Document Conventions
This manual uses the following typographic conventions:
Example
Description
This icon calls attention to important safety notes.
Warning!
A Warning indicates the potential for bodily harm and
tells you how to avoid the problem.
Caution
A Caution indicates potential damage to the instrument
and tells you how to avoid the problem.
Note
Bold text is primarily used for emphasis.
This icon calls attention to important information.
Intended Use Statement
The Synergy 2 is a single-channel multi-mode microplate reader. The performance
characteristics of the data reduction software have not been established with any
laboratory diagnostic assay. The user must evaluate this instrument and PC-based
software in conjunction with the specific assay(s). This evaluation must include the
confirmation that performance characteristics for the specific assay(s) are met.
BioTek Gen5 software package provides the user with instrument control and data
reduction capabilities.
The Synergy 2 can operate with standard robotic systems, such the BioStack Microplate
Stacker.
This product may be used for In Vitro Diagnostic, research and development, or other
non-clinical purposes.
Quality Control
It is considered good laboratory practice to run laboratory samples according to
instructions and specific recommendations included in the assay package insert for the test
to be conducted. Failure to conduct Quality Control checks could result in erroneous test
data.
Synergy 2 Operator’s Manual
12 | Preface
Warranty and Product Registration
Please review the Warranty information that shipped with your product. Register your
product with BioTek to ensure that you receive important information and updates.
Register online through the Customer Resource Center (CRC) at www.biotek.com or by
calling (888) 451-5171 or (802) 655-4740.
Warnings
Operate the instrument on a level surface away from excessive humidity.
Strong light can reduce the linear performance range of the instrument.
Measurement values may be affected by extraneous particles (such as dust) in the
microplate wells. A clean work area is necessary to ensure accurate readings.
When operated in a safe environment according to the instructions in this
document, there are no known hazards associated with the instrument. However,
the operator should be aware of certain situations that could result in serious injury;
these may vary depending on the instrument model. See Hazards and Precautions.
Hazards
The following hazard warnings are provided to help avoid injury:
Warning! Power Rating. The instrument’s power supply cord must be connected
to a power receptacle that provides voltage and current within the specified rating
for the system. Use of an incompatible power receptacle may produce electrical
shock and fire hazards.
Warning! Electrical Grounding. Never use a two-prong plug adapter to connect
primary power to the external power supply. Use of an adapter disconnects the
utility ground, creating a severe shock hazard. Always connect the power supply
directly to an appropriate receptacle with a functional ground.
Warning! Internal Voltage. Always turn off the power switch and unplug the
power supply before cleaning the outer surface of the instrument.
Warning! Liquids. Avoid spilling liquids on the reader; fluid seepage into internal
components creates a potential for shock hazard or instrument damage. If a spill
occurs while a program is running, abort the program and turn off the instrument.
Wipe up all spills immediately. Do not operate the instrument if internal
components have been exposed to fluid.
BioTek Instruments, Inc.
Precautions |
13
Warning! Unspecified Use. Failure to operate this equipment according to the
guidelines and safeguards specified in this manual could result in a hazardous
condition.
Warning! Software Quality Control. The operator must follow the
manufacturer’s assay package insert when modifying software parameters and
establishing reading methods. Failure to conduct quality control checks could result
in erroneous test data.
Warning! Reader Data Reduction Protocol. No limits are applied to the raw
absorbance data. All information exported via computer control must be thoroughly
analyzed by the operator.
Warning! Hot Surface. The tungsten lamp assembly is hot when the instrument is
turned on. Turn off the reader and allow the lamp to cool down before attempting to
replace it.
Warning! Pinch Hazard. Some areas of the Dispense Module can present pinch
hazards when the instrument is operating. These areas are marked with the symbol
shown in the Safety Symbols section of this Preface. Keep hands/fingers clear of
these areas when the instrument is operating.
Warning! Potential Biohazards. Some assays or specimens may pose a
biohazard. Adequate safety precautions should be taken as outlined in the assay’s
package insert. Always wear safety glasses and appropriate protective equipment,
such as chemically resistant rubber gloves and an apron.
Precautions
The following precautions are provided to help avoid damage to the instrument:
Caution: Service. The instrument should be serviced by BioTek authorized service
personnel. Only qualified technical personnel should perform troubleshooting and
service procedures on internal components.
Caution: Environmental Conditions. Do not expose the system to temperature
extremes. For proper operation, ambient temperatures should remain within the
range listed in the Specifications section of Chapter 1. Performance may be
adversely affected if temperatures fluctuate above or below this range. Storage
temperature limits are broader.
Caution: Sodium Hypochlorite. Do not expose any part of the instrument to the
recommended diluted sodium hypochlorite solution (bleach) for more than 20
minutes. Prolonged contact may damage the instrument surfaces. Be certain to rinse
and thoroughly wipe all surfaces.
Caution: External Power Supply. Only use the power supply shipped with the
instrument. Operate this power supply within the range of line voltages listed on it.
Synergy 2 Operator’s Manual
14 | Preface
Caution: Shipping Panel/Hardware. The shipping panel, carrier shipping
screw, optic arm shipping block, and mirror holder shipping bracket must be
removed before operating the instrument. They must be reinstalled before
repackaging the reader for shipment. See Chapter 2, Installation.
Caution: Disposal. This instrument contains printed circuit boards and wiring
with lead solder. Dispose of the instrument according to Directive 2002/96/EC, “on
waste electrical and electronic equipment (WEEE)” or local ordinances.
Caution: Warranty. Failure to follow preventive maintenance protocols may void
the warranty. See Chapter 6 for preventive maintenance procedures.
Caution: Electromagnetic Environment. Per IEC 61326-2-6 it is the user’s
responsibility to ensure that a compatible electromagnetic environment for this
instrument is provided and maintained in order that the device will perform as
intended.
Caution: Electromagnetic Compatibility. Do not use this device in close
proximity to sources of strong electromagnetic radiation (e.g., unshielded
intentional RF sources), because these may interfere with the proper operation.
CE Mark
Based on the programs described below and information contained herein,
this product bears the CE mark.
™ See the Declaration of Conformity for more information.
Directive 89/336/EC: Electromagnetic Compatibility
Emissions - Class A
The system has been type tested by an independent, accredited testing laboratory and
found to meet the requirements of EN 61326-1 and EN61326-2-6: Class A for Radiated
Emissions and Line Conducted Emissions.
Verification of compliance was conducted to the limits and methods of EN 55011 –
(CISPR 11) Class A. In a domestic environment it may cause radio interference, in
which case you may need to mitigate the interference.
Immunity
The system has been type tested by an independent, accredited testing laboratory and
found to meet the requirements of EN 61326-1 and EN 61326-2-6 for Immunity.
Verification of compliance was conducted to the limits and methods of the following:
EN 61000-4-2 Electrostatic Discharge
EN 61000-4-3 Radiated EM Fields
EN 61000-4-4 Electrical Fast Transient/Burst
BioTek Instruments, Inc.
CE Mark |
15
EN 61000-4-5 Surge Immunity
EN 61000-4-6 Conducted Disturbances from RFI
EN 61000-4-11 Voltage Dips, Short Interruptions and Variations
Directive 73/23/EEC Low Voltage (Safety)
The system has been type tested by an independent testing laboratory and was found
to meet the requirements of EC Directive 73/23/EEC for Low Voltage. Verification of
compliance was conducted to the limits and methods of the following:
EN 61010-1:2001, “Safety requirement for electrical equipment for measurement,
control and laboratory use. Part 1, General requirements.”
EN 61010-2-81:2003, “Requirements for automatic and semi-automatic laboratory
equipment for analysis and other purposes.”
EN 61010-101:2002, “Particular requirements for in vitro diagnostic (IVD) medical
equipment.”
Directive 2002/96/EC: Waste Electrical and Electronic Equipment
Disposal Notice: This instrument contains printed circuit boards and wiring with lead
solder. Dispose of the instrument according to Directive 2002/96/EC, “on waste
electrical and electronic equipment (WEEE)” or local ordinances.
Directive 98/79/EC: In Vitro Diagnostics
•
Product registration with competent authorities
•
Traceability to the U.S. National Institute of Standards and Technology (NIST)
Synergy 2 Operator’s Manual
16 | Preface
Electromagnetic Interference and Susceptibility
USA FCC CLASS A
Warning: Changes or modifications to this unit not expressly approved by the
manufacturer could void the user's authority to operate the equipment.
This equipment has been tested and found to comply with the limits for a Class A digital
device, pursuant to Part 15 of the FCC Rules.
These limits are designed to provide reasonable protection against harmful interference
when the equipment is operated in a commercial environment. Like all similar equipment,
this equipment generates, uses, and can radiate radio frequency energy and, if not
installed and used in accordance with the instruction manual, may cause harmful
interference to radio communications. Operation of this equipment in a residential area is
likely to cause interference, in which case the user will be required to correct the
interference at his own expense.
Canadian Department of Communications Class A
This digital apparatus does not exceed Class A limits for radio emissions from digital
apparatus set out in the Radio Interference Regulations of the Canadian Department of
Communications.
Le present appareil numerique n'met pas du bruits radioelectriques depassant les limites
applicables aux appareils numerique de la Class A prescrites dans le Reglement sur le
brouillage radioelectrique edicte par le ministere des Communications du Canada.
User Safety
This device has been type tested by an independent laboratory and found to meet the
requirements of the following:
•
Underwriters Laboratories UL 61010-1: 2004, “Safety requirements for electrical
equipment for measurement, control and laboratory use; Part 1: general
requirements”
•
Canadian Standards Association CAN/CSA C22.2 No. 61010-1-04 , “Safety
requirements for electrical equipment for measurement, control and laboratory
use; Part 1: general requirements”
•
EN 61010 Standards – See CE Mark list
BioTek Instruments, Inc.
Safety Symbols |
17
Safety Symbols
Some of these symbols appear on the instrument or accessories:
Alternating current
Courant alternatif
Wechselstrom
Corriente alterna
Corrente alternata
Both direct and alternating current
Courant continu et courant alternatif
Gleich - und Wechselstrom
Corriente continua y corriente alterna
Corrente continua e corrente alternata
Direct current
Courant continu
Gleichstrom
Corriente continua
Corrente continua
Earth ground terminal
Borne de terre
Erde (Betriebserde)
Borne de tierra
Terra (di funzionamento)
On (Supply)
Marche (alimentation)
Ein (Verbindung mit dem
Netz)
Conectado
Chiuso
Protective conductor terminal
Borne de terre de protection
Schutzleiteranschluss
Borne de tierra de protección
Terra di protezione
Off (Supply)
Arrêt (alimentation)
Aus (Trennung vom Netz)
Desconectado
Aperto (sconnessione dalla rete
di alimentazione)
Caution (refer to accompanying
documents)
Attention (voir documents
d’accompanement)
Achtung siehe Begleitpapiere
Atención (vease los documentos incluidos)
Attenzione, consultare la doc annessa
Warning, risk of electric shock
Attention, risque de choc
électrique
Gefährliche elektrische schlag
Precaución, riesgo de sacudida
eléctrica
Attenzione, rischio di scossa
elettrica
Warning, risk of crushing or pinching
Attention, risque d’écrasement et
pincement
Warnen, Gefahr des Zerquetschens und
Klemmen
Precaución, riesgo del machacamiento y
sejeción
Attenzione, rischio di schiacciare ed
intrappolarsi
Warning, hot surface
Attention, surface chaude
Warnen, heiße Oberfläche
Precaución, superficie caliente
Attenzione, superficie calda
Warning, potential biohazards
Attention, risques biologiques potentiels
Warnung! Moegliche biologische Giftstoffe
Atención, riesgos biológicos
Attenzione, rischio biologico
Synergy 2 Operator’s Manual
18 | Preface
In vitro diagnostic medical
device
Dispositif médical de
diagnostic in vitro
Medizinisches In-VitroDiagnostikum
Dispositivo médico de
diagnóstico in vitro
Dispositivo medico
diagnostico in vitro
Separate collection for electrical and
electronic equipment
Les équipements électriques et
électroniques font l’objet d’une collecte
sélective
Getrennte Sammlung von Elektro- und
Elektronikgeräten
Recogida selectiva de aparatos eléctricos y
electrónicos
Raccolta separata delle apparecchiature
elettriche ed elettroniche
Consult instructions for use
Consulter la notice d’emploi
Gebrauchsanweisung beachten
Consultar las instrucciones de
uso
Consultare le istruzioni per
uso
BioTek Instruments, Inc.
Chapter 1
Introduction
This chapter introduces the Synergy 2, describes its key features,
and lists its package contents. Page 24 contains information on
contacting BioTek for product support and service.
Product Description ..................................................................20
Package Contents ....................................................................21
Optional Accessories ................................................................22
Product Support and Service .....................................................24
20 | Chapter 1: Introduction
Product Description
Depending on the model, Synergy 2 detection modes include Fluorescence Intensity (FI),
Fluorescence Polarization (FP), Time-Resolved Fluorescence (TRF), Luminescence and UVvisible Absorbance. The Synergy 2 is a single-channel microplate reader for research and
development and in vitro diagnostic use.
Synergy 2 uses a unique combination of monochromator, filters and dichroic mirrors. Its
three broad-spectrum light sources have been chosen for optimal illumination and
excitation in all applications. The reader is computer-controlled using BioTek’s Gen5 PC
software for all operations including data reduction and analysis. The Synergy 2 is robot
accessible and compatible with BioTek’s BioStack Microplate Stacker. Gen5 supports OLE
automation to facilitate the Synergy 2’s integration into an automated system.
Two light sources are available for Fluorescence determinations , a tungsten halogen lamp
(part of the FI and FP modules) or a xenon flash (part of the TRF module) along with
interference filters and dichroic mirrors for wavelength specificity, and a photomultiplier
tube (PMT) detector. The Synergy 2 has both top and bottom probes for fluorescence
measurements. Bottom probe readings do not use the mirrors. The Synergy 2 models with
Fluorescence Polarization (FP) capability are equipped with polarizing filters.
Luminescence is measured by the low-noise PMT detector through an empty filter position
in the Emission filter wheel. Filters can also be used if light filtering is necessary.
Absorbance measurements are made by switching to a super-quiet xenon flash lamp and a
monochromator for wavelength selection. The xenon lamp allows for both UV and visible
light absorbance measurements. The monochromator provides wavelength selection from
200 to 999 nm in 1-nm increments. Area and spectral scanning, and Pathlength Correction
are available read methods.
The Synergy 2 has a 4-Zone™ temperature control from 4°C over ambient to 50°C. Internal
plate shaking is supported to ensure that reagents are properly mixed prior to reading.
The Synergy 2 supports the reading of 6-, 12-, 24-, 48-, 96-, 384-, and 1536-well microplates
with standard 128 x 86 mm geometry, as well as the BioTek Take3 Multi-Volume Plate.
Note: The Luminescence system does not support the reading of 1536-well plates.
Models with injectors support dual-reagent dispensing to 6-, 12-, 24-, 48-, and 96-well
microplates. An external dispense module pumps fluid from the supply bottles to the two
injectors located inside the instrument. Both injectors are positioned directly above the
bottom probe, and fluid is injected into one well at a time.
BioTek Instruments, Inc.
Package Contents |
Package Contents
™ Part numbers and package contents are subject to change. Contact
BioTek Customer Care with any questions.
Item
Part #
Synergy 2 Operator’s Manual
7131000
Power supply
7130560
Power cord set (specific to installation environment):
Europe (Schuko)
USA/International
United Kingdom
Australia/New Zealand
75010
75011
75012
75013
RS-232 serial cable
75034
USB cable with Virtual COM Driver Software
Wrench
75108
7090204
48576
Filter “plugs” (2) (also referred to as “dummy filters” or “blanks”)
7082073
Spare filter retaining clips (2)
7082075
Plastic storage bag and Velcro strips
Models with injectors (“D” dispenser models), an external dispense
module (packed separately), with the following accessories:
Outlet tubes (2, plus 2 spare) from dispense module to instrument
Inlet tubes (2) from supply bottles to syringe drives
250 µl syringes (2)
Syringe thumbscrews (2)
Priming plate
Injector tip priming trough
Dispense module communication cable
Dispense module front cover
Dispense module box
Supply bottles (2, 30 ml)
Supply bottle holder assemblies (2)
Injector tip cleaning stylus and plastic storage bag
Synergy 2 Operator’s Manual
—
7082120
7082121
7083000
19511
7132158
7092133
75107
7082137
7090568
7122609
7090564
2872304
21
22 | Chapter 1: Introduction
Optional Accessories
™ Part numbers and accessories are subject to change. Contact BioTek
Customer Care with any questions or visit www.biotek.com and use
the Accessories search tool.
Item
Part #
Synergy 2 Product Qualification (IQ-OQ-PQ) package
7130012
Test Plate for absorbance measurement testing
7260522
Harta Luminometer Reference Microplate and Adapter
8030015
Glowell Adapter Plate
7160006
Take3 Multi-Volume Plate
TAKE3
BioCell Quartz Vessel
BioCell Adapter Plate
7272051
7270512
PCR Tube Adapter Plates
6002072
6002076
Terasaki Adapter Plate
7330531
UV-Range (300 nm and above) Excitation Polarizer
7132041
3-foot (1 meter) external Dispense Module tubing
7112186
Empty filter wheel
7080541
Filter retainer clip
7082075
Filter wheel plug (dummy filter)
7082073
Empty mirror holder
7130564
Empty mirror holder, FP-compatible
7130563
Replacement Tungsten Lamp
7080500
Replacement Shipping Materials
7130016
Additional Filters, Filter Wheels, Mirrors, and Mirror Holders; contact BioTek for part
numbers and availability.
The Synergy 2 is compatible with the BioStack Microplate Stacker. Contact BioTek or
visit our website to learn more.
For Use with Liquid Tests (see Chapter 5)
Absorbance Liquid Test Solutions:
BioTek Wetting Agent Solution
BioTek QC Check Solution #1
25 mL
125 mL
Dispense Module Liquid Test Solution:
BioTek Green Test Dye
Part #
7773002
7120779
7120782
7773003
BioTek Instruments, Inc.
Optional Accessories |
For Use with Liquid Tests (see Chapter 5)
Fluorescence Test Kits (includes microplates and solutions):
Complete Kit (Sodium Fluorescein, Europium, Methylumbelliferone)
Sodium Fluorescein Kit
Fluorescence Polarization Kit
TRF Europium Kit
Methylumbelliferone “MUB” Kit
Individual Fluorescence Liquid Test Solutions:
Sodium Fluorescein Powder
10 mg vial of Methylumbelliferone (“MUB”)
Carbonate-Bicarbonate Buffer (“CBB”) capsules
Part #
7160010
7160013
7160014
7160011
7160012
98155
98156
98158
Fluorescence Polarization Liquid Test Solutions are also available from Invitrogen
Corporation, in their “FP One-Step Reference Kit,” part number P3088.
The Time-Resolved Fluorescence Liquid Test Solution is also available from
Invitrogen Corporation: FluoSpheres carboxylate-modified microspheres, 0.2 µm
europium luminescent, 2 mL, PN F20881.
Synergy 2 Operator’s Manual
23
24 | Chapter 1: Introduction
Product Support and Service
Technical Assistance Center (TAC)
If your instrument or software fails to function properly, if you have questions about
how to use or maintain our products, or if you need to send an instrument to BioTek
for service or repair, please contact our Technical Assistance Center (“TAC”).
The TAC is open from 8:30 AM to 5:30 PM (EST), Monday through Friday, excluding
standard U.S. holidays.
Phone: (800) 242-4685 or
Fax: (802) 654-0638
(802) 655-4740
E-Mail: tac@biotek.com
Web: www.biotek.com
Please be prepared to provide the following information:
•
Your name and company information, along with a daytime phone or fax
number, and/or an e-mail address
•
The product name, model, and serial number
•
The onboard software part number and basecode version (in Gen5, select
System > Reader Control > Information)
•
For troubleshooting assistance or instruments needing repair, the specific steps
that produce your problem and any error codes displayed in Gen5 (see also
Appendix B, Error Codes)
If you need to return an instrument to BioTek for service or repair, please contact the
TAC for a Return Materials Authorization (RMA) number and the shipping address.
See page 43 for repackaging instructions.
Applications Support
BioTek’s fully equipped Application Laboratory provides our on-staff scientists with
the means to assist you with the integration of our instrumentation and software with
your unique scientific applications. If you are having difficulty with optimizing
fluorescence sensitivity or integrating a unique data reduction transformation, or you
are just looking for a recommendation on an appropriate fluorophore, contact us.
Phone: (888) 451-5171
E-Mail: applications@biotek.com
BioTek Instruments, Inc.
Chapter 2
Installation
This chapter includes instructions for unpacking and setting up the
reader and its components.
Product Registration.................................................................26
Important Information..............................................................26
1: Unpack and Inspect the Reader .............................................27
2: Unpack and Inspect the Dispense Module ................................28
3: Select an Appropriate Location ..............................................29
4: Remove the Shipping Hardware .............................................29
5: Install the Power Supply .......................................................34
6: Install the Dispense Module...................................................35
7: Connect the Host Computer ..................................................37
8: Install Gen5 on the Host Computer.........................................38
9: Turn on the Power Supply and Reader ....................................38
10: Establish Communication ....................................................39
11: Run a System Test .............................................................39
12: Test the Injector System.....................................................41
Operational/Performance Qualification ........................................42
Repackaging and Shipping Instructions .......................................43
26 | Chapter 2: Installation
Product Registration
If you have not already done so, please register your product(s) with BioTek to ensure that
you receive important information and updates about the products you have purchased.
Register online through the BioTek Customer Resource Center at www.biotek.com or by
contacting BioTek Customer Care (see page 8).
Important Information
This chapter contains installation and setups tasks for a Synergy 2 reader
configured with all of the available modules (the “SLFPTAD” model).
Your Synergy 2 model may be different. For example, it may not have
injection capability, or it may not have an Excitation filter wheel (“SL”
and “SLD” models). Perform the installation and setup tasks in the order
presented, skipping those that do not apply to your reader’s
configuration.
Materials: You will need a slotted screwdriver and a Phillips-head
screwdriver to perform some of the steps in this section. You will also
need a small wrench; this item is supplied with the instrument.
Remove the shipping hardware before turning on the instrument.
Re-install the shipping hardware and attach the shipping panel before
repackaging the instrument for shipment.
BioTek Instruments, Inc.
1: Unpack and Inspect the Reader |
27
1: Unpack and Inspect the Reader
The reader should be removed from the box by two people. It weighs up to
57 pounds (25.8 kg), depending on the model.
Save all packaging materials. If you need to ship the reader to BioTek for
repair or replacement, you must use the original materials. Using other
forms of commercially available packaging, or failing to follow the
repackaging instructions, may void your warranty.
During the unpacking process, inspect the packaging, reader, and
accessories for shipping damage. If the reader is damaged, notify the
carrier and your BioTek representative. Keep the shipping boxes and the
packaging materials for the carrier’s inspection. BioTek will arrange for
repair or replacement of your reader immediately.
1. Set the outer shipping box close to the intended work surface. Open the outer
shipping box and remove the foam blocks to access the inner shipping box.
2. Open the inner shipping box. Remove the accessories that are stored inside the
cardboard shipping insert and then remove the insert.
3. The reader is attached to a shipping panel, which has two handles for lifting (see
Figure 1 on page 28). With one person on each side, locate and grasp the handles.
Carefully lift the reader out of the box. Place the reader on its back on the work
surface, so the reader lies flat and the panel hangs over the edge of the surface.
4. Using a slotted screwdriver, remove the screws and washers that attach the panel to
the reader. Carefully set the reader upright.
5. Place the panel with the screws and washers into the shipping box for storage. Place
the packaging materials in the shipping boxes for reuse if the reader needs to be
shipped again.
Synergy 2 Operator’s Manual
28 | Chapter 2: Installation
Figure 1: The reader on its back with the shipping panel attached
to the bottom (left), and removing the screws (right).
2: Unpack and Inspect the Dispense Module
Save all packaging materials. If you need to ship the dispense module to
BioTek for repair or replacement, you must use the original materials.
Using other forms of commercially available packaging, or failing to
follow the repackaging instructions, may void your warranty.
During the unpacking process, inspect the packaging, module, and
accessories for shipping damage. If the module is damaged, notify the
carrier and your BioTek representative. Keep the shipping boxes and the
packing materials for the carrier’s inspection. BioTek will arrange for
repair or replacement of your dispense module immediately.
1. Open the outer shipping box. Remove the foam cap, inner shipping box, and
accessories box.
2. Open the inner shipping box. Remove the two reagent bottle holders and the
cardboard shipping insert. Lift out the module and place it on a level surface.
3. Open the accessories box and remove its contents. The accessories should include the
dispense module-related items on page 21.
4. Place all packaging materials into the shipping box for reuse if the dispense module
needs to be shipped again.
BioTek Instruments, Inc.
3: Select an Appropriate Location |
29
3: Select an Appropriate Location
Install the reader on a level surface in an area where ambient temperatures between 18°C
(64°F) and 40°C (104°F) can be maintained. The reader is sensitive to extreme
environmental conditions. Avoid the following:
•
Excessive humidity. Condensation directly on the sensitive electronic circuits can
cause the instrument to fail internal self-checks. The humidity must be in the range
of 10-85%, non-condensing.
•
Excessive ambient light. Bright light may affect the reader’s optics and readings,
reducing its linear range.
•
Dust. Readings may be affected by extraneous particles (such as dust) in the
microplate wells. A clean work area is necessary to ensure accurate readings.
4: Remove the Shipping Hardware
Caution: Remove all shipping hardware before you turn the reader on.
1. Locate the two screws that secure the shroud; one on each side of the reader, in the
lower rear corners. Remove the screws (Figure 2) and slide off the shroud (Figure 3).
Remove two screws, one
on each side of the reader
Figure 2: Removing the two side screws.
Synergy 2 Operator’s Manual
30 | Chapter 2: Installation
Figure 3: Sliding off the shroud.
2. Identify the areas inside the instrument where the shipping hardware is currently
installed, and where some items will be stored:
Optic arm
vertical knob
Mirror holder
shipping bracket
Storage
Optic arm
shipping bracket
Microplate carrier
shipping screw and tube
Figure 4: Internal compartment, with shipping hardware.
BioTek Instruments, Inc.
4: Remove the Shipping Hardware |
31
3. Use the supplied wrench to remove the microplate carrier shipping screw. You will
store the screw on the base plate as shown in Figure 7. Pull the flexible carrier
shipping tube off of the carrier. Store the tube in the plastic storage pocket. Use the
supplied Velcro strips to attach the pocket to the back of the reader for storage.
Tube
Figure 5: Remove microplate carrier shipping screw and tube.
4. Remove three screws
(with washers) that
hold the optic arm
shipping block in
place. Turn the knob
at the top of the
motor clockwise to
raise the optic arm.
Slide the block out
from under the arm.
Knob; turn
clockwise after
removing screws
Screws (3)
Shipping block
Figure 6: Removing the optic arm shipping block
Synergy 2 Operator’s Manual
32 | Chapter 2: Installation
5. Store the optic arm shipping block and three screws/washers on the base plate.
Shipping screw
Figure 7: Carrier shipping screw and optic arm shipping
block stored on the base plate.
6. Remove the two screws that secure the mirror holder shipping bracket.
Figure 8: Removing the mirror holder shipping bracket.
BioTek Instruments, Inc.
4: Remove the Shipping Hardware |
7. Turn the bracket over and replace the screws.
Figure 9: The mirror holder shipping bracket
in its storage position.
8. Slide the shroud onto the reader and replace the two side screws (Figure 3 on
page 30).
Synergy 2 Operator’s Manual
33
34 | Chapter 2: Installation
5: Install the Power Supply
Warning! Power Rating. The instrument must be connected to a
power receptacle that provides voltage and current within the specified
rating for the system. Use of an incompatible power receptacle may
produce electrical shock and fire hazards.
Warning! Electrical Grounding. Never use a plug adapter to connect
primary power to the instrument. Use of an adapter disconnects the
utility ground, creating a severe shock hazard. Always connect the
system power cord directly to an appropriate receptacle with a
functional ground.
Perform these steps to install the power supply:
1. Locate the power inlet on the right side
of the reader, it is labeled “POWER IN.”
2. Examine the power supply’s plug. It has
a small groove that will line up with a
tab inside the power inlet. The plug is
encircled by a securing ring.
3. Insert the plug into the power inlet.
Align the groove with the tab and twist
the ring clockwise until the plug is firmly
seated.
4. Plug the power supply’s cord into an
appropriate power receptacle.
Power
inlet
Figure 10: The external power supply (left) and the reader’s power inlet (right).
BioTek Instruments, Inc.
6: Install the Dispense Module |
35
6: Install the Dispense Module
1. Place the dispense module on top of the reader.
Figure 11: The dispense module on top of the reader.
2. On the rear panel of the reader, identify the SYRINGE 1 and SYRINGE 2 tubing
ports. Remove the nylon screws from both ports.
3. Open two of the plastic bags containing the outlet tubes. Remove the clear plastic
fitting shrouds from the tubes. Put the other two bags in a safe place; they are spares.
4. Place the nylon screws and the plastic fitting shrouds in the plastic tool storage bag.
Use the supplied Velcro strips to attach the bag to the rear panel of the dispense
module.
5. Remove the two inlet tubes from their protective plastic canisters.
6. Identify the two circular syringe valves on the dispense module. Each is labeled with
a left-pointing arrow.
Synergy 2 Operator’s Manual
36 | Chapter 2: Installation
™ When installing the inlet and outlet tubes, do not use any tools.
Finger-tighten only!
7. Screw the fitting of one inlet tube into the right side of the Syringe 1 valve.
8. Screw one end of one outlet tube into the left side of the Syringe 1 valve.
9. Screw the other end of the outlet tube into the SYRINGE 1 port on the rear of the
reader.
10. Repeat these steps to attach the inlet and outlet tubing for Syringe 2.
11. Seat the outlet tubes in the clip to the left of the Syringe 2 valve.
Outlet
tubes
Inlet
tubes
Syringe
valves
Figure 12: The dispense module’s outlet and inlet tubes, and syringe valves.
12. Remove the two syringes from their protective boxes. They are identical and
interchangeable. Each syringe should already be assembled, but if there are two
separate pieces, assemble them now: insert the white tip of the syringe plunger into
the barrel of the syringe and gently push it all the way into the barrel.
13. Install both syringes, referring to Figure 13.
•
Hold the syringe vertically with the threaded end at the top and the
knurled steel end at the bottom.
•
Screw the threaded end of the syringe into the bottom of the syringe valve.
Finger-tighten only.
BioTek Instruments, Inc.
7: Connect the Host Computer |
37
•
Carefully pull down the knurled steel end of the syringe until it is resting
inside the hole in the bracket.
•
Pass a metal thumbscrew up through this hole and thread it into the bottom
of the syringe. Hold the syringe from rotating while tightening the
thumbscrew. Finger-tighten only.
Syringes
Syringe
brackets
Thumbscrews
Figure 13: The dispense module; close-up view of the syringes.
14. Locate the dispense module cable. Plug one end into the port on the left side of the
dispense module. Plug the other end into the “Dispenser Port” on the right side of
the reader.
15. Locate the injector tip-cleaning stylus, packaged in a small plastic cylinder. Attach
the cylinder to the back of the dispense module for storage.
7: Connect the Host Computer
The Synergy 2 is equipped with two types of communication ports: Serial (RS-232) and
USB.
•
Both types of cables are included in the accessories box. Determine which cable is
supported by the host computer.
•
Connect one end to the appropriate port on the reader and the other end to the
appropriate port on the host computer.
Synergy 2 Operator’s Manual
38 | Chapter 2: Installation
8: Install Gen5 on the Host Computer
The Synergy 2 is controlled by Gen5 software running on a host computer.
There is a certain sequence of events that must be followed to ensure that
the software is properly installed and configured. Please follow the
instructions provided in the Gen5 Getting Started Guide to install the
software.
9: Turn on the Power Supply and Reader
1. If Gen5 is open, close it now.
2. The power supply has its own switch. Locate the switch and turn the power supply on.
3. The reader’s power switch is located on the lower left corner of the front panel. Locate
the switch and turn the reader on. The reader will perform a System Test. When the test
is complete, the microplate carrier will eject.
Carrier eject
button
Power
switch
Power switch
Figure 14: The reader with its microplate carrier extended.
™ The carrier eject button, located above the reader’s power switch, is
used to extend/retract the microplate carrier.
BioTek Instruments, Inc.
10: Establish Communication |
39
10: Establish Communication
1. If you are using the USB cable, refer to the instructions that shipped with the USB Driver
Software CD to install the necessary drivers.
2. Start Gen5 and log in if prompted.
™ The default System Administrator password is admin.
3. Open the System Menu and select Reader Configuration.
4. Click Add and set the Reader Type to Synergy 2.
5. Set the Com Port to the computer’s COM port to which the reader is attached.
6. Click Test Comm. Gen5 attempts to communicate with the reader. If the attempt is
successful, return to the Gen5 main screen.
Communication Errors
•
Is the reader connected to the power supply and turned on?
•
Is the communication cable firmly attached at both ends?
•
Did you select the correct Reader Type in Gen5?
•
Try a different Com Port.
•
If using the USB cable, did you install the driver software?
•
If you remain unable to get Gen5 and the reader to communicate with each other,
contact BioTek’s Technical Assistance Center.
11: Run a System Test
Running a System Test will confirm that the reader is set up and running properly, or will
provide an error code if a problem has been detected.
1. Turn on the incubator:
•
In Gen5, select System > Reader Control > Synergy 2.
•
Click the Pre-Heating tab.
•
Enter a Requested temperature of at least 37°C and click On.
•
Return to Gen5’s main view.
Synergy 2 Operator’s Manual
40 | Chapter 2: Installation
2. If applicable, adjust Gen5’s Absorbance Wavelengths table to values that will
confirm operation of the reader at its limits. We recommend 200 and 999 nm (the
lower and upper limits of the monochromator), and any four wavelengths in
between that best represent your assays and/or the lowest and highest wavelength
values typically used in your lab.
•
Select System > Reader Configuration. Highlight the Synergy 2 reader
and click View/Modify.
•
Click Setup and then click the Absorbance tab. The six wavelength values
currently in use are displayed. (You may wish to make a note of the current
values, so you can reset them later.)
•
Enter the six wavelength values you wish to test and then click Send
Wavelengths to download them to the reader.
™ Wait until the incubator temperature reaches the set point before continuing.
3. Select System > Diagnostics > Run System Test. If prompted to select a reader,
select the Synergy 2 and click OK.
4. When the test is complete, a dialog will appear to request additional information.
Enter the information and click OK.
5. The results report will appear. Scroll down toward the bottom, the text should read
“SYSTEM TEST PASS.”
•
You may wish to print the report and store it with your Installation records.
•
The software stores system test information in its database; you can retrieve
it at any time.
™ If an error code is returned, refer to Appendix B and look up the
code. If the problem is something you can fix, do so now and run
another System Test. If the problem is something you cannot fix, or if
the test continues to fail, contact BioTek’s Technical Assistance Center.
6. Turn off the incubator:
•
Select System > Reader Control > Synergy 2.
•
Click the Pre-Heating tab and click Off.
7. Models with injectors: Keep Gen5 open and proceed to the next section.
All other models: The installation and setup process is complete. Close Gen5 and
proceed to Operational/Performance Qualification on page 42.
BioTek Instruments, Inc.
12: Test the Injector System |
41
12: Test the Injector System
1. If necessary, press the carrier eject button to eject the microplate carrier.
2. Place the tip priming trough in the left rear pocket of the carrier.
3. Place the priming plate on the carrier.
Tip priming
trough
Priming plate
Figure 15: Priming trough and plate installed on the carrier.
4. Fill the two reagent bottles with distilled or deionized water. Place the bottles in
their holders, and place the holders directly in front of the syringes. Insert the inlet
tubes into the bottles.
5. In Gen5, select System > Reader Control > Synergy 2 (Com<#>)
6. Click the Dispenser tab.
7. With Dispenser set to 1, set the Volume to 5000 µL and click Prime.
The syringe should move down and up repeatedly, drawing fluid from the bottle.
The fluid should pump through the tubing and dispense into the priming plate.
Examine the fittings; no leaks should be detected. If leaks are detected, tighten all
fittings and repeat the prime. If leaks are still detected, contact BioTek TAC.
8. When the prime finishes, set Volume to 2000 µL and click Purge to clear the fluid
lines.
9. Set Dispenser to 2 and repeat steps 7 and 8.
10. When finished, remove and empty the priming plate. Close the software.
Synergy 2 Operator’s Manual
42 | Chapter 2: Installation
Operational/Performance Qualification
Your Synergy 2 reader was fully tested at BioTek prior to shipment and should operate
properly following the successful completion of the installation and setup procedures
described throughout this chapter.
If you suspect that problems occurred during shipment, if you received the reader back
from BioTek following service or repair, and/or if regulatory requirements dictate that
Operational/Performance Qualification is necessary, turn to Chapter 5, Instrument
Qualification now to learn about BioTek’s recommended OQ/PQ procedures for the
Synergy 2.
™ A Product Qualification & Maintenance (IQ/OQ/PQ) package for the
Synergy 2 is available for purchase (PN 7130566). Contact your local
BioTek dealer for more information.
BioTek Instruments, Inc.
Repackaging and Shipping Instructions |
Repackaging and Shipping Instructions
Important! Please read the information provided below
before preparing the Synergy 2 for shipment.
If the reader and/or dispense module has been exposed to potentially
hazardous material, decontaminate it to minimize the risk to all who
come in contact with the reader during shipping, handling and servicing.
Decontamination prior to shipping is required by the U.S. Department of
Transportation regulations. See Chapter 7, As Needed Maintenance for
decontamination instructions.
Remove the microplate and tip prime trough (if equipped) from the
carrier before shipment. Spilled fluids can contaminate the optics and
damage the instrument.
The instrument’s packaging design is subject to change. If the
instructions in this section do not apply to the packaging materials you
are using, please contact BioTek’s Technical Assistance Center for
guidance.
Replace the shipping hardware before repackaging the reader. Please
contact BioTek if you have misplaced any of these items, and order PN
7130016:
•
Carrier shipping tube
•
Carrier shipping screw/o-ring
•
Optic arm shipping block/screws
•
Mirror holder shipping bracket/screws
If you need to ship the reader and/or the dispense module to BioTek for
service or repair, be sure to use the original packaging materials. Other
forms of commercially available packaging are not recommended and
can void the warranty.
The shipping materials are designed to be used no more than five times.
If the original materials have been damaged, lost, or used more than five
times, contact BioTek to order replacements:
•
PN 7133005, shipping container for the Synergy 2 reader
•
PN 7083001, shipping container for the dispense module
Synergy 2 Operator’s Manual
43
44 | Chapter 2: Installation
Perform these steps to prepare the reader for shipment:
1. Contact BioTek’s Technical Assistance Center for an RMA (Return Materials
Authorization) number and the shipping address. See page 24 for contact
information.
2. Decontaminate the reader and, if attached, the dispense module, according to the
instructions provided in Chapter 7.
3. If you will also be shipping the dispense module, perform these steps:
•
If you have not already done so as a part of decontamination, purge the
dispense module and fluid lines.
•
With the reader on, start Gen5 and select System > Reader Control >
Synergy 2 (Com<#>).
•
Perform this step twice, for both dispensers: Click the Dispenser tab and
set the dispenser number (1 or 2). Click Maintenance. The syringe bracket
will lower. Remove the thumbscrew from underneath the bracket. Carefully
unscrew the top of the syringe from the syringe valve. Lift out the syringe
and store it in its original box.
•
Fully detach the dispense module from the reader. Replace the two nylon
screws into the Syringe 1 and 2 tubing ports on the rear of the reader. (The
screws should be stored in the plastic bag attached to the back of the
module.) Set the module aside for the moment.
4. If applicable, remove the tip priming trough and store it in the accessories bag.
5. If you have not already done so, retract the microplate carrier and then turn off and
unplug the reader.
6. Remove the shroud and replace the shipping hardware (see page 29). Replace the
shroud when finished.
7. Carefully tip the reader onto its back. Attach the shipping panel to the bottom of the
reader using the four flat-head screws and washers (see page 27).
8. Locate the original outer shipping box. Place four foam blocks in the four bottom
corners of the box. Place the inner shipping box inside the outer box.
9. Using two people, grasp the handles on the shipping panel and carefully lower the
reader into the inner shipping box.
10. Slide the cardboard insert straight down into place around the reader. Place the
accessories inside the pockets in the insert.
11. Close and seal the inner box with tape.
12. Place four foam corner blocks around the inner shipping box. Close and seal the
outer box with tape.
13. Write the RMA number in large clear numbers on the outside of the box. Ship the
box to BioTek.
BioTek Instruments, Inc.
Repackaging and Shipping Instructions |
45
Perform these steps to prepare the dispense module for shipment:
1. If you have not already done so:
•
Contact BioTek’s Technical Assistance Center for an RMA (Return Materials
Authorization) number and the shipping address. See page 24 for contact
information.
•
Decontaminate the module according to the instructions in Chapter 7. Be
sure to purge the dispense module of all fluid when finished.
•
Remove the two syringes (see step 3 on the previous page) and store them
in their original boxes.
•
Detach the dispense module outlet tubes and communication cable from the
reader. Replace the two nylon screws into the Syringe 1 and 2 tubing ports
on the rear of the reader.
™ Refer to the figures on the next pages when performing these steps
2. Remove the two inlet tubes from the syringe valves and store them in their plastic
canisters.
3. Remove the two outlet tubes from the syringe valves. Attach the clear plastic fitting
shrouds to the fittings of the outlet tubes. Place the tubes in a plastic bag.
4. Place the dispense module inside the inner shipping box. Slide the cardboard
shipping insert down around the module. Pack the reagent bottle holders in bubble
wrap and place them on top of the module. Seal the box with tape.
5. Locate the original accessories shipping box and foam end caps. Place the bottom
foam end cap into the box.
6. Place the syringes, the inlet tubes, and the outlet tubes inside the cutouts of the
bottom foam end cap in the accessories box. Place the dispense module shroud on
top of the accessories.
7. Shroud the accessories with the top foam end cap, place the dispense module cable
inside the top of the end cap, and seal the box with tape.
8. Locate the original outer shipping box and foam end caps. Insert the bottom foam
end cap. Lower the dispense module box into the end cap.
9. Insert the accessories box alongside the dispense module box.
10. Insert the top foam end cap. Close and seal the outer box with tape.
11. Write the RMA number in large, clear numbers on the outside of the box. Ship the
box to BioTek.
Synergy 2 Operator’s Manual
46 | Chapter 2: Installation
Dispense module
cable
Top foam
end cap
Dispense
module
shroud
Inlet tubes (2)
Syringes (2)
Outlet tubes (4)
Bottom foam
end cap (with
cutouts)
Accessories
box
Figure 16: Dispense module accessories.
BioTek Instruments, Inc.
Repackaging and Shipping Instructions |
Reagent bottle
holders (2)
Cardboard
Shipping
insertinsert
Dispense
module
Dispense module
Inner
Inner
shipping box
shipping box
Top foam
end cap
Inner shipping box
(dispense module)
Accessories
box
Bottom foam
end cap
Outer
shipping box
Figure 17: The contents of the inner (top) and outer (bottom) shipping boxes.
Synergy 2 Operator’s Manual
47
48 | Chapter 2: Installation
BioTek Instruments, Inc.
Chapter 3
Getting Started
This chapter describes some of the Synergy 2’s main external and
internal components, and provides an introduction to using Gen5
software to control the instrument.
Modular Design .......................................................................50
External Components ...............................................................51
Internal Components................................................................52
Tungsten Lamp.....................................................................53
Excitation/Emission Filters......................................................54
Mirrors ................................................................................55
Injector System.......................................................................56
Gen5 Software ........................................................................59
Define Excitation/Emission Filters ............................................59
Define Mirrors ......................................................................59
Protocols and Experiments .....................................................60
Dispense Module Control........................................................61
Recommendations for Optimum Performance...............................62
50 | Chapter 3: Getting Started
Modular Design
The Synergy 2 is a multi-mode microplate reader, with a design that allows you to initially
purchase only the module(s) you need and then upgrade later as your experimental
requirements expand. For example, if you initially purchased an “SLFTA” model you can
add the Fluorescence Polarization and/or Dispense modules. Please contact BioTek
Customer Care to learn more about your upgrade options.
The table below briefly describes the available modules. The module letters form the part
number for each Synergy 2 model; for example, a reader with all modules installed is an
SLFPTAD. This is indicated on a label on the back of the reader.
Module
Description
S
Synergy 2 base model. Includes incubation control, shaking, and Gen5
software.
L
Luminescence
F
Fluorescence Intensity, top and bottom. Includes the TungstenHalogen light source.
P
Fluorescence Polarization. Requires the “F” module.
T
Time-Resolved Fluorescence. Requires “F” module. Includes the high
energy DPR Xenon Flash Lamp.
A
UV-Visible Absorbance (monochromator-based). Includes the SQ
Xenon flash lamp.
D
Dual Reagent Dispenser.
BioTek’s Gen5 software is used to control all of the reader’s functions. If the reader is
connected and turned on, Gen5 will present you only with options that apply to your
reader model. For example, if your model is not equipped with the Dispense module,
Gen5 will not provide the option for you to add a Dispense step to your assay protocol.
BioTek Instruments, Inc.
External Components |
External Components
The dispense module (if equipped)
sits on top of the reader.
ALWAYS PURGE the lines before
disconnecting the tubing.
The side panel contains
the communication and
dispenser ports, the input
for the power supply, and
a green light that indicates
the power supply is ON.
The light-blocking
microplate carrier
access door.
Press this button
to eject/retract the
microplate carrier,
or to stop the
reader from
beeping when an
error has occurred.
The power supply, which
has its own power switch.
The power switch, with a
green light that indicates
the reader is ON.
The access door for filter
wheels and the Tungsten
lamp (if equipped).
Figure 18: Identifying the main external components
Synergy 2 Operator’s Manual
51
52 | Chapter 3: Getting Started
Internal Components
This section introduces some internal components that may require replacement and/or
cleaning over time. Each topic provides references to other sections of this manual for
more detailed information and instructions. As discussed on page 50, not all of these
components exist in all Synergy 2 models.
Component
What you need to know about it
Page
Tungsten Lamp
Requires replacement after approximately 1000
hours of use. Applies to models with the
Fluorescence Intensity and/or Fluorescence
Polarization modules.
53
Excitation and
Emission Filters
The filters and filters wheels can be changed to
accommodate your assays, and filters should be
cleaned periodically. Applies to models with the
Fluorescence Intensity, Fluorescence Polarization,
Time-Resolved Fluorescence, and/or Luminescence
modules.
54
Mirrors
The mirrors and mirror holder can be changed to
accommodate your fluorescence assays. Mirrors may
require cleaning over time. Applies to models with
the filter-based Fluorescence Intensity, TimeResolved Fluorescence and Fluorescence Polarization
modules.
55
Injector System
The syringes may require replacement over time.
The tubing and internal reading chamber may
require cleaning over time. Applies to models with
the Dispense module.
56
BioTek Instruments, Inc.
Internal Components |
53
Tungsten Lamp
Description and Location
Synergy 2 models with the Fluorescence Intensity and/or Fluorescence
Polarization modules are equipped with a Tungsten lamp. The lamp is accessed
through a hinged door on the front of the instrument. To open the door, press on
its lower left and right corners until the door opens downward. The lamp is on the
left, behind a light shield with a hot surface warning label.
The Tungsten lamp is
behind this shield
Figure 19: Locating the Tungsten lamp
When to Replace the Lamp
The Tungsten lamp is expected to operate without replacement for a minimum of
1000 hours. The intensity of the bulb will slowly drop over time until the reader’s
System Test detects a low signal and displays an error message. In addition, error
code 2901 may be displayed during normal operation. The lamp should be
replaced at this time; contact BioTek and order part number 7080500.
See Chapter 7, As Needed Maintenance for replacement instructions.
Keep the front door closed during operation. The intense
broad spectrum light of the xenon lamp can cause eye
damage. The light shield shown above mitigates the risk.
Synergy 2 Operator’s Manual
54 | Chapter 3: Getting Started
Excitation/Emission Filters
Synergy 2 models with the Fluorescence Intensity module are equipped with
Excitation and Emission filter wheels. (Models with the Luminescence module without
Fluorescence Intensity have an Emission filter wheel only.) The filter wheels are
labeled as “EX” or “EM” and are accessed through a hinged door on the front of the
instrument. To open the door, press on its lower left and right corners until the door
opens downward.
Excitation
filter wheel
Emission
filter wheel
Thumbscrews
for removal
Diagram showing
orientation of glass
filters in filter wheels
Figure 20: Locating the Excitation and Emission filter wheels
Each filter wheel contains four equal-sized positions. Each position can contain a glass
filter or a light-blocking plug, or can be left empty. Filter wheel contents can be
changed, as well as entire filter wheels.
Gen5 keeps track of each wheel’s contents, and communicates this information to the
instrument during operation. If you make any changes to either filter wheel, you must
update Gen5’s filter table (System > Reader Configuration).
See Chapter 4, Filters and Mirrors for information on working with the filter wheels,
and for a list of filters available from BioTek.
EX/EM Configuration for Luminescence
•
For best results when taking luminescence measurements, the Excitation filter
wheel (if equipped) should have no empty locations.
•
If your tests require that the light emitted from the samples remain unfiltered,
the Emission filter wheel should have an empty location in it. When selecting a
filter set for a read in Gen5, selecting ‘Hole’ indicates the empty location.
BioTek Instruments, Inc.
Internal Components |
55
Mirrors
When taking fluorescence (FI, FP, or TRF) measurements from the top, the Synergy 2
uses mirrors to direct the excitation and emission light paths. The mirrors are stored in
a mirror holder, which is a rectangular box located inside the reader. The holder stores
up to three mirrors. There are two possible mirror types:
•
A 50% mirror is a glass slide with silver dots (see #1 below). It works with any
wavelength in the range of 200 to 850 nm.
•
A dichroic mirror is wavelength specific; it requires the excitation and
emission filters to fall within specific ranges. Dichroic mirrors provide better
sensitivity than 50% mirrors, but they are dye-specific.
The default mirror configuration is shown below (any changes should be reflected in
your sales order). A 50% mirror is easily identified by its “dots,” and each dichroic
mirror is etched or labeled with its wavelength value.
#3
510 nm dichroic
#2
400 nm dichroic
#1
50%
Polarizing filters
Figure 21: Identifying the mirrors in the mirror holder (default configuration shown).
Models with the Fluorescence Polarization module also have EX polarizing
filters in the mirror holder, as shown inside the oval above.
The entire mirror holder and the individual mirrors are user-changeable. Because the
mirrors can be easily smudged or even damaged, however, BioTek strongly
recommends changing the entire mirror holder. Contact BioTek for more information
on purchasing additional mirrors and holders.
To learn more about how mirrors are used, and how to change or clean them, see
Chapter 4, Filters and Mirrors.
Synergy 2 Operator’s Manual
56 | Chapter 3: Getting Started
Injector System
External Dispense Module
™ If a syringe is leaking, it may need to be replaced. See Chapter 7, As
Needed Maintenance for instructions.
Each dispense module is calibrated to perform with a specific
Synergy 2 reader. Make sure the same serial number appears
on both the dispense module and the reader.
The dispense module pumps fluid from the supply bottles to injector heads located
inside the instrument. Fluid is injected into one well at a time.
e
f
c
d
Figure 22: Dispense module components
BioTek Instruments, Inc.
Injector System |
57
1
Two 250-µL syringes draw fluid from the supply bottles.
2
Inlet tubes transport fluid from the supply vessels to the syringes. These tubes are short
pieces of opaque PTFE (Teflon) tubing connected to stainless steel probes on one end and
threaded fittings on the other end.
3
Three-way valves switch the syringe flow from the inlet tubes to the outlet tubes.
4
Outlet tubes transport fluid from the syringes into the instrument, through the tubing
ports on the reader’s rear panel. The outlet tubes are opaque PTFE tubes with threaded
fittings on each end that are used to deliver fluid from the syringes to the instrument.
Internal Tubing
Inside the reader, two Teflon tubes transport fluid from the tubing ports on the rear of
the instrument to the two injectors. As shown below, both injectors are positioned
directly above the bottom fluorescence optical probe.
Syringe 1
Syringe 2
Bottom probe
Figure 23: Injectors inside the reader
™ The tubing and injectors should be cleaned at least quarterly. See
Chapter 6, Preventive Maintenance for more information.
Synergy 2 Operator’s Manual
58 | Chapter 3: Getting Started
Priming the Injector System
Before running a Dispense assay, prime the system with the reagent or dispensing
fluid. Additionally, tip priming can be performed at the start of the assay and
sometimes, just before each dispense to a well. The tip prime compensates for any fluid
loss at the injector tip due to evaporation since the last dispense. All priming activities
are controlled via Gen5.
Both types of primes require a fluid reservoir to be present on the microplate carrier:
•
The priming plate is placed on the microplate carrier for a Prime operation (to
prime the dispense system with fluid).
•
The tip priming trough is a small, removable cup located in the left rear of the
carrier, and is used for performing the Tip Prime before dispensing. The trough
holds up to 1.5 mL of liquid and must be periodically emptied and cleaned by
the user.
™ Do not perform Tip Priming when using tall plates. Generally,
plates with fewer than 96 wells are too tall for error-free tip priming;
and, tip priming is rarely required for these larger-volume plates.
Tip priming
trough
Priming plate
Figure 24: The tip priming trough and priming plate
installed on the microplate carrier
BioTek Instruments, Inc.
Gen5 Software |
59
Gen5 Software
BioTek Gen5 software supports all Synergy 2 reader models. Use Gen5 to control the
reader and the dispense module (if equipped), perform data reduction and analysis on the
measurement values, print /export results, and more.
This section provides brief instructions for working with Gen5 to define the contents of
filter wheels and mirror holders, create protocols and experiments, and read plates. Refer
to the Gen5 Help system for more information.
Define Excitation/Emission Filters
For models equipped with Excitation and Emission filter wheels, the reader’s onboard
software is configured with the filter values and their locations in the filter wheels.
When Gen5 communicates with the reader, it requests this information and stores it in
a filter table on the computer. If you make any changes to the filter wheels, you must
define the changes in Gen5 and send the information to the reader.
To access the filter wheel information in Gen5:
•
Select System > Reader Configuration. Highlight the Synergy 2 reader, and
click View/Modify.
•
Click Setup and then click the Fluorescence/Luminescence or
Absorbance tab. Refer to the Gen5 help system for additional instructions.
Define Mirrors
For models equipped with a mirror holder, the reader’s onboard software is
configured with the mirror types and their characteristics. When Gen5 communicates
with the reader, it requests this information and stores it in a mirror table on the
computer. If you change any mirrors, you must define the changes in Gen5 and send
the information to the reader.
To access the mirror holder information in Gen5:
•
Select System > Reader Configuration. Highlight the Synergy 2 reader, and
click View/Modify.
•
Click Setup and then click the Mirrors tab. Refer to the Gen5 help system for
additional instructions.
Synergy 2 Operator’s Manual
60 | Chapter 3: Getting Started
Protocols and Experiments
In Gen5, a Protocol contains instructions for controlling the reader and (optionally)
instructions for analyzing the data retrieved from the reader. At a minimum, a protocol
specifies the Procedure for the assay you wish to run. After creating a protocol, you
can create an Experiment that references the protocol. You can then run the
experiment to read plates and analyze the data.
The instructions below briefly describe how to create a simple protocol in Gen5. See
the Gen5 Help system for complete instructions.
1. Select File > New Protocol.
2. Open the Procedure dialog. If prompted to select a reader, select the Synergy 2
and click OK.
3. Add steps to the procedure for shaking or heating the plate, dispensing fluid,
reading the plate, and more. Click Validate to verify that the reader supports the
defined steps, and then click OK.
Optionally, perform the next steps to analyze and report the results:
4. Open the Plate Layout dialog and assign blanks, samples, controls, and/or
standards to the plate.
5. Open the Data Reduction dialog to add data reduction steps. Categories include
Transformation, Well Analysis, Curve Analysis, Cutoff, Fluorescence
Polarization and Validation.
6. Create a report or export template, via the Report Builder, File Export Builder, or
Power Export Builder options.
7. Select File > Save and give the protocol file an identifying name.
The instructions below briefly describe how to create a simple experiment and then
read a plate in Gen5. See the Gen5 Help system for complete instructions.
1. Select File > New Experiment.
2. Select the desired protocol and click OK.
3. Highlight a plate in the menu tree and click Read. The Plate Reading dialog will
appear.
4. Click READ. The door will open and the carrier will extend (if it is not already
extended).
5. Place the plate on the carrier and click OK to begin the read.
6. When the read is complete, measurement values will appear in Gen5. Use the
Plate View to view the results. (Double-click a plate in the menu tree to open the
Plate View.) Select the desired data set (e.g., “528/20,645/40”) from the Data list.
7. Select File > Save and give the experiment file an identifying name.
BioTek Instruments, Inc.
Gen5 Software |
61
Dispense Module Control
This section only applies to models with injectors.
Gen5 is used to perform several dispense module-specific functions, such as initialize,
dispense, prime, and purge. The Prime and Purge functions are introduced here. See
the Gen5 help system for more information.
™ Priming and purging routines are used to clean the fluid path; see also
“Flush/Purge the Fluid Path” on page 143.
Prime
Before running a procedure with a Dispense step, prime the system with the fluid
to be used:
1. Important! Place the priming plate on the carrier.
2. Fill the supply bottle with a sufficient volume of the fluid to be used for the
prime and the assay. Insert the appropriate inlet tube into the bottle.
3. In Gen5, select System > Reader Control > Synergy 2 (Com<#>) and
click the Dispenser tab.
4. Select the Dispenser number (1 or 2) associated with the supply bottle.
5. Enter the Volume to be used for the prime. The minimum recommended
prime volume is 1000 µL.
6. Select a prime Rate, in µL/second.
7. Click Prime to start the process.
8. When finished, carefully remove the priming plate from the carrier and
empty it.
™ If the priming plate is empty, the prime volume was too low.
Purge
To save reagent, Gen5 provides a method to purge fluid from the system back into
the supply bottle:
1. In Gen5, select System > Reader Control>Synergy 2 (Com<#>) and
click the Dispenser tab.
2. Select the Dispenser number (1 or 2) associated with the supply bottle.
3. Enter the desired purge Volume in µL.
4. Select a prime Rate in µL/second.
5. Click Purge to start the process.
Synergy 2 Operator’s Manual
62 | Chapter 3: Getting Started
Recommendations for Optimum Performance
Microplates should be clean and free of dust or bottom scratches. Use new microplates
from sealed packages. Do not allow dust to settle on the surface of the solution; use
microplate covers or seals when not reading the plate. Filter solutions to remove
particulates that could cause erroneous readings.
Before preparing your microplates, make sure the reader is on and communicating with
Gen5. You may want to run a System Test if the reader has not been turned off/on in a few
days. Design your Gen5 protocol in advance as well, to ensure that the intended reading
parameters are used and to avoid any last-minute corrections.
Although the Synergy 2 supports standard flat, U-bottom, and V-bottom microplates, the
reader achieves optimum performance with flat-bottomed wells. See Appendix A
Specifications for information on supported plates.
Non uniformity in the optical density of the well bottoms can cause loss of accuracy,
especially with U- and V-bottom polyvinyl microplates. Check for this by reading an
empty microplate. Dual wavelength readings can eliminate this problem, or bring the
variation in density readings to within acceptable limits for most measurements.
Inaccuracy in pipetting has a large effect on measurements, especially if smaller volumes
of liquid are used. For best results, use at least 100 µL per well in a 96-well plate, 25 µL in a
384-well plate and 5 µL in a 1536-well plate.
Pipetting solution into 384- and 1536-well plates often traps air bubbles in the wells, which
may result in inaccurate readings. A dual-wavelength reading method usually eliminates
these inaccuracies. However, for best results, remove the air bubbles by degassing the
plate in a vacuum chamber or spinning the plate in a centrifuge before reading.
The inclination of the meniscus can cause loss of accuracy in some solutions, especially
with small volumes. Agitate the microplate before reading to help bring this problem
within acceptable limits. Use Tween® 20, if possible (or other wetting agent) to normalize
the meniscus for absorbance measurements. Some solutions develop menisci over a period
of several minutes. This effect varies with the brand of microplate and the solution
composition. As the center of the meniscus drops and shortens the light path, the density
readings change. The meniscus shape will stabilize over time.
To keep the dispense system in top condition, flush and purge the fluid lines with
deionized (DI) water every day or upon completion of an assay run, whichever is more
frequent. Some reagents may crystallize or harden after use, clogging the fluid
passageways. Flushing the tubing at the end of each day, letting the DI water soak them
overnight, and then purging the lines at the beginning of each day ensures optimal
performance of the dispense system. See Chapter 6, Preventive Maintenance for more
information.
Models with injectors: When dispensing volumes less than or equal to 20 µL/well, BioTek
recommends specifying a tip prime volume equal to the dispense volume. For dispense
volumes greater than 20 µL/well, BioTek recommends a tip prime volume of 20 µL.
BioTek Instruments, Inc.
Chapter 4
Filters and Mirrors
Chapter 3 provided an overview of the filters and mirrors installed
in Synergy 2 models with the Fluorescence Intensity module. This
chapter provides more detailed information on working with these
components.
Excitation/Emission Filters ........................................................64
Change the Filter Wheels and Filters ........................................65
Clean the Filters ...................................................................66
Filters Available from BioTek ..................................................67
Mirrors ...................................................................................68
Change the Mirror Holder and Mirrors ......................................71
Clean the Mirrors ..................................................................76
Mirrors Available from BioTek .................................................77
64 | Chapter 4: Filters and Mirrors
Excitation/Emission Filters
The Synergy 2 is equipped with filter wheels for use with obtaining fluorescence and
luminescence measurements. The Excitation filter selects the band of light to which the
sample will be exposed. The Emission filter selects the band of light with the maximum
fluorescence signal of the sample, to be measured by the photomultiplier tube (PMT).
The filter wheels are accessed by opening the hinged door on the front of the reader.
Figure 25: Accessing the Excitation (EX) and Emission (EM) filter wheels
Each filter wheel is labeled as EX or EM, and can contain up to four filters and/or black
plugs (also referred to as “dummy filters”). Each filter has its central wavelength and band
pass values printed on its side, with an arrow to indicate the proper direction of light
through the filter (see Figure 26). Filters and plugs are secured with C-clip retainers.
Figure 26: EX and EM filter wheels, showing correct orientation of the filters
BioTek Instruments, Inc.
Excitation/Emission Filters |
65
Change the Filter Wheels and Filters
The filter wheels are easily exchanged to meet your assay requirements. If you
regularly need to change the filters on the reader, consider purchasing additional filter
wheels from BioTek to make the process easier and faster.
As shown in Figure 25, labels on the front of the filter wheels can be marked with the
central wavelength and band pass of each filter.
When removing/replacing filter wheels:
•
It is critical that the Gen5 Filter Table matches the actual filter locations in the
EX and EM filter wheels. See “Define Excitation/Emission Filters” on page 59.
•
The Excitation and Emission filter wheels are not interchangeable and are
labeled EX = Excitation, EM = Emission.
•
Filter direction within each filter wheel is important, and the direction differs
depending on the filter wheel type. A diagram on the inside of the reader’s
front panel door indicates orientation; see Figure 26.
•
Each filter is marked with an arrow indicating the correct direction of light
through the filter. Filters are not specific to either excitation or emission.
To remove a filter wheel:
1. Using your thumbs, push down on the bottom corners of the hinged door on the
front of the reader to open the door.
2. Observe the two thumbscrews within the compartment. The left thumb-screw
secures the Excitation filter wheel; the right secures the Emission filter wheel.
3. Remove the thumbscrew and slide the filter wheel’s supporting metal bracket
straight out of the compartment.
™ The Emission filter wheel “springs” out when removed (a shutter
behind the wheel closes quickly to protect the PMT).
To install a filter wheel:
1. Ensure that all filters and/or plugs are inserted properly (see Figure 26).
2. Slide the filter wheel into its chamber.
3. Replace the thumbscrew and close the front door.
4. Use Gen5 to update the reader’s internal software with the current filter wheel
configuration; see “Define Excitation/Emission Filters” on page 59.
Synergy 2 Operator’s Manual
66 | Chapter 4: Filters and Mirrors
When removing or replacing a filter or C-clip retainer, do not use a sharp
object. Use several layers of lens paper and your finger to remove and
replace filters and clips. Using a sharp object, such as a flat screwdriver,
will scratch the filter surface and make it unusable. Do not touch the
filters with your bare fingers.
To remove a filter or plug:
1. Remove the filter wheel as instructed on the previous page.
2. Turn the filter wheel to align the desired filter with the hole in the supporting
bracket.
3. Place the bracket on a flat surface, with the filter wheel facing down.
4. Prepare a multi-layered “cushion” of lens paper. Using your finger covered with
the lens paper, gently push against the filter and its retainer until they pop out.
To replace a filter or plug:
1. Hold the metal bracket with the filter wheel facing up.
2. Orient the filter or plug (see Figure 26): Observe the arrow on the filter indicating
the light direction. Align the filter’s wavelength number with the window in
each filter holder, then drop it into the desired location.
™ Make note of the filter position number, 1–4.
3. Using your fingers, squeeze the sides of the C-clip retainer, and then insert it into
the top of the hole containing the new filter. Cover your finger with several
layers of lens paper, and then push down on all sides of the retainer until it sits
flush against the filter.
4. Gently wipe both sides of the filter with lens paper.
5. When finished, install the filter wheel.
Clean the Filters
Instructions are provided in Chapter 6, Preventive Maintenance.
BioTek Instruments, Inc.
Excitation/Emission Filters |
67
Filters Available from BioTek
Bandpass filters are available for purchase from BioTek. Please note that part numbers
are subject to change, and new filters may become available. Custom filters are also
available. Contact BioTek Customer Care with any questions.
PN
7082259
7082248
7082250
7082263
7082254
7082230
7082220
7082228
7082242
7082205
7082206
7082227
7082207
7082208
7082222
7082221
7082209
7082256
7082218
7082246
7082247
7082223
7082249
7082253
7082210
7082215
7082211
7082212
7082264
7082245
7082244
7082225
7082224
7082252
7082265
7082251
7082213
7082214
7082257
7082266
7082226
7082229
Wavelength
284/10
310/20
320/20
330/80
340/11
340/30
360/40
380/20
400/10
400/30
420/50
440/30
440/40
450/50
460/40
485/20
485/40
500/27
508/20
516/20
528/20
530/25
540/25
540/35
545/40
560/15
560/20
560/40
570/100
575/15
580/50
590/20
590/35
600/40
620/10
620/15
620/40
635/32
645/15
665/7.5
645/40
680/30
Main Application
Tryptophan excitation
Tyrosine emission, O-aminobenzoyl excitation
7-methoxycoumarin and Quanta Blu excitation
HTRF excitation
Fura-2 excitation
NADH excitation and tryptophan emission
MUB, caspace-3, europium chelate excitation
Fura-2 and EBFP excitation
Porphyrin excitation, O- aminobenzoyl and 7-methoxycoumarin emission
CFP excitation and Quanta-Blu emission
Attophos excitation and caspace-3 emission
NADH emission
CBQCA excitation
NanoOrange excitation and EBFP and MUB emission
Fluorescein, EGFP excitation and CFP emission
Propidium Iodide excitation
YFP excitation
Fura-2 emission
EGFP emission
VIC excitation and Fluorescein and EGFP emission
5-Tamra excitation
Alexa Fluor 546, CY3, and rhod2 excitation and EYFP emission
Alamar Blu, Amplex red, RFP excitation
Rhodamine B excitation
Cell Titer Blue excitation
VIC emission
Attophos and CBQCA emission
AlphaScreen emission
ROX excitation and CY3 and 5-Tamra emission
NanoOrange and Attophos emission
Alexa Fluor 594 and Texas Red excitation and Cell Titer Blue emission
Rhod-2, Alexa Fluor 546, and CY3 emission
Alamar Blu, Amplex Red, RFP and porphyrin emission
HTRF / LANCE emission
ROX and Alexa Fluor 594 emission and Alexa Fluor 633 excitation
Rhodamine B, europium chelate emission, CY5 excitation
Texas Red emission
Alexa Fluor 633 emission
HTRF / LANCE emission
Texas Red and Propidium iodide emission
CY5 emission, AlphaScreen excitation
The fluorescence ratio associated with the HTRF readout is a correction method developed
by CIS bio and covered by the US patent 5,527,684 and its foreign equivalents, for which
CIS bio has granted a license to BioTek. Its application is strictly limited to the use of HTRF
reagents and technology, excluding any other TR-FRET technologies.
Synergy 2 Operator’s Manual
68 | Chapter 4: Filters and Mirrors
Mirrors
™ See also “Mirrors” on page 55.
For top-reading fluorescence analysis, the Synergy 2 uses mirrors to direct the excitation
and emission light paths. Mirrors are required for fluorescence polarization (FP)
measurements to direct light to the sample, because fibers cannot carry polarized light.
Mirrors also provide increased sensitivity for fluorescence intensity (FI) and time-resolved
fluorescence (TRF) measurements when compared to mapped fiber optics.
Mirrors are stored in a three-mirror holder (additional mirror holders and mirrors can be
purchased as accessories). The holder and individual mirrors can be changed to meet your
requirements. You can replace the entire holder with a different one; this is the BioTek
recommended option. Alternatively, you can install different mirrors in the holder.
Contact BioTek for more information on purchasing mirrors and holders.
For Synergy 2 models with the FP module, the reader is equipped with three polarizers:
•
Excitation polarizer (visible-range or UV-range, see page 70)
•
Emission polarizer, parallel to excitation polarizer
•
Emission polarizer, perpendicular to excitation polarizer
Inside the reader, the mirror holder is labeled with five position numbers (see Figure 27 on
the next page), which translate to three possible measurement positions (see Figure 28).
The third measurement position is dedicated to FP, because it holds the polarizers. It is
twice the size of positions 1 and 2, and it is numbered 3, 4, 5 in the reader. Gen5 recognizes
only the three measurement positions. FI and TRF can be performed using position 3 as
well. See Figure 29 for a close-up view of measurement position 3.
When running an experiment, Gen5 communicates with the reader to move the holder to
the proper position based on the mirror you define in a Read step in the Gen5 protocol.
BioTek Instruments, Inc.
Mirrors |
5 f
3
d
c
Figure 27: Mirror holder removed from the reader
UV Block
3 mirrors
=
3 reading
positions
Position 3 holds the polarizers
Figure 28: Mirror holder diagram; the three reading positions are indicated
Synergy 2 Operator’s Manual
69
70 | Chapter 4: Filters and Mirrors
™ Position 1 in the mirror holder has an emission UV-blocking filter. It
transmits light above 380 nm and blocks light below 360 nm. If you
are using a 50% mirror and an excitation above 360 nm, for best
results place the 50% mirror in position 1 (the default location). If
emission of your assay is below 380 nm, do not use position 1 for
these measurements; move the 50% mirror to position 2.
™ Models with the FP module: The polarizers are always installed in
positions 4 and 5, which translates to Mirror #3 in Gen5.
EM Parallel
EM Perpendicular
This diagram shows Position 3 in the
mirror holder. It holds the polarizer
filters required for Fluorescence
Polarization.
Open
Positions
It is the only mirror position that can be
selected for an FP read.
It can also be selected for FI and TRF,
also, which use the open positions.
Mirror
EX Polarizer
Figure 29: Mirror holder; Position 3 diagram
Two types of excitation (EX) polarizers are available: visible-range (400 nm and above, the
default) or UV-range (300 nm and above, available from BioTek). The visible-range
polarizer’s bracket is etched with “400 nm”. The emission (EM) polarizers are installed
inside the reader, in the mirror holder’s top plate.
The default mirror holder configuration is described here; your reader may be configured
differently. Turn to page 77 for a list of mirrors available from BioTek.
Position
#
Mirror
#
Mirror
Type
Polarizers
EX Range
(nm)
EM Range
(nm)
Use
1
1
50%
None
200–850
200–850
FI, TRF
2
2
400 nm
Dichroic
None
320–390
410–800
FI, TRF
3
4
5
None
3
510 nm
Dichroic
EX (visible-range),
parallel EM
EX (visible-range),
perpendicular EM
FI, TRF
440–505
515–640
FP
BioTek Instruments, Inc.
Mirrors |
71
Change the Mirror Holder and Mirrors
™ Do not touch the mirrors. These optical elements are delicate and
must be handled carefully. The glass and anti-reflective (AR) coated
surfaces are damaged by any contact, especially by abrasive particles.
Wear cloth gloves to reduce the risk of damaging the mirrors and
polarizing filters. For cleaning instructions see Chapter 7, Preventive
Maintenance.
Because dichroic mirrors are wavelength specific, it may be necessary to change the
mirror holder before performing certain assays. BioTek offers additional mirror
holders and mirrors as separate accessories.
Mirrors and polarizing filters are easily damaged. If more than three unique mirrors
are used in your lab, the preferred method is to use multiple mirror holders, which can
be exchanged as needed. BioTek offers additional mirror holders and mirrors as
separate accessories.
Removing the mirror holder from the reader is required in either scenario. Before
removing the holder, take a moment to identify the reader’s components that are
described in the following procedures.
Mirror holder in position for removal
Mirror
holder
top
plate
The mirror holder is
attached to the top
plate from below.
The top plate shows
numbers for each
position in the
mirror holder and
holds the EM
polarizer filters in
FP models.
Mirror holder’s belt drive
Figure 30: Mirror holder positioned for removal
Synergy 2 Operator’s Manual
72 | Chapter 4: Filters and Mirrors
To remove the mirror holder
1. Turn off the reader and remove its shroud (see instructions on page 147).
2. The mirror holder will likely be stored in its “home” position; you’ll need to
move it to an accessible position. Use the holder’s belt drive (see below) to roll
the holder (to the left) away from the optics armature. Use your thumb to turn
the belt counterclockwise.
Figure 31: Turn the belt drive to position the holder for removal
3. When the mirror holder is fully exposed, carefully remove the two screws and
washers on top, and set them aside. The holder will continue to be held in place
by its magnetized frame.
4. Grasping the sides of the holder underneath the top plate, pull the holder down
and toward the back of the reader to remove it.
Note: The light shield is the
black metal plate on the
back of the mirror holder
Figure 32: Mirror holder removed from the reader
BioTek Instruments, Inc.
Mirrors |
73
5. When you change the holder, update Gen5 with the new mirror configuration:
•
Select System > Reader Configuration.
•
Double-click the Synergy 2 reader to open the Reader Settings dialog.
•
Click Setup.
•
Select the Mirrors tab.
•
For each Mirror position, 1, 2, 3, use the drop-down list to select the
Type of mirror. For dichroic mirrors, enter the excitation and emission
wavelength ranges. (Refer to the Gen5 help for more information.)
™ It is critical the Gen5 mirror table reflects the actual location and
characteristics of the mirrors in the reader.
™ If you accidentally touch the mirrors or polarizing filters, see Chapter
7, Preventive Maintenance for cleaning instructions.
To change a mirror in the mirror holder
Use these tools to change a mirror:
•
Linen or cloth gloves
•
Small Phillips screwdriver
™ Touch the mirrors as little as possible; hold them by their edges only.
The mirrors are easily damaged.
1. Remove the mirror holder from the reader.
2. The holder has a light shield to protect the mirrors (Figure 33). Use a Phillips
screwdriver to remove the four screws and washers that attach this shield to the
holder.
3. Use the Phillips screwdriver to remove the bracket that secures the mirrors
(Figure 34). Set aside the screws. Lift the bracket away from the holder and set
aside.
4. Grasping the mirror by its edges, remove it from the holder and store it properly.
5. Holding the replacement mirror by its edges, turn the mirror so its label is faceup and readable. Align it in this orientation when you insert it into the holder.
6. Replace the metal bracket to secure the mirror. The bend in the bracket’s arms
should point away from the holder.
7. When you change the mirrors, update Gen5 with the new mirror configuration.
Synergy 2 Operator’s Manual
74 | Chapter 4: Filters and Mirrors
™ It is critical that the Gen5 Mirror Table reflects the actual location and
characteristics of the mirrors in the reader.
™ If you accidentally touch the mirrors or polarizing filters, see Chapter
7, Preventive Maintenance for cleaning instructions.
™ The light shield only fits the holder one way. Check its alignment when
reattaching it to the mirror holder.
Figure 33: Remove the light shield from the mirror holder
BioTek Instruments, Inc.
Mirrors |
Mirror label
Figure 34: Mirror orientation; labels are face-up and readable
The bend in the bracket’s arm
points away from the holder
Figure 35: Mirror holder with the light shield removed
Synergy 2 Operator’s Manual
75
76 | Chapter 4: Filters and Mirrors
To reinstall the mirror holder
1. Hold the mirror holder with the alignment pins on top and the filter windows
facing you. This is the holder’s orientation inside the reader.
2. Inside the reader, put the mirror holder underneath its top plate (see Figure 30).
The holder’s alignment pins will help guide it into place and because it is
magnetized the holder will stay in place.
3. Reinstall the two sets of screws and washers that secure it in the reader.
Alignment pins
Filter windows
Figure 36: Mirror holder positioned for reinstallation
Clean the Mirrors
Instructions for cleaning the mirrors and polarizers (if equipped) are provided in
Chapter 7, Preventive Maintenance.
BioTek Instruments, Inc.
Mirrors |
77
Mirrors Available from BioTek
Please note that part numbers are subject to change, and new mirrors may become
available. Contact BioTek Customer Care with any questions.
•
Half-Size mirrors fit into positions 1 and 2 of the mirror holder
•
Full-Size mirrors fit into position 3.
Half-Size
Part #
Full-Size
Part #
Cut-off
(nm)
Excitation
Range
Emission
Range
7132121
n/a
50%
200–850
200–850
All except FP
7138365
n/a
365
290–350
380–800
HTRF, MMP, Quanta Blu
7138400
7137400
400
320–390
410–800
MUB, Europium, Hoechst
33258
7138455
7137455
455
400–450
460–710
Attophos, CFP, Fluo-3
7138510
7137510
510
440–505
515–640
Fluorescein, Picogreen,
FAM
7138525
7137525
525
475–520
530–670
Rhodamine 123, YFP
7138550
7137550
550
415–540
560–850
CY3, HEX, Rhodamine 6G
7138570
7137570
570
515–565
575–735
Alamar Blu, Amplex Red,
TAMRA
7138595
7137595
595
540–590
600–770
ROX, Texas Red
7139635
n/a
635
640–780
400–630
AlphaScreen
7138660
7137660
660
580–655
665–850
CY5
Synergy 2 Operator’s Manual
Main Applications
78 | Chapter 4: Filters and Mirrors
BioTek Instruments, Inc.
Chapter 5
Instrument Qualification
This chapter contains procedures for qualifying the initial and
ongoing performance of the Synergy 2 and the external dispense
module (if used).
Overview ................................................................................80
IQ/OQ/PQ...............................................................................80
Recommended Qualification Schedule .........................................82
System Test............................................................................83
Absorbance Plate Test ..............................................................87
Absorbance Liquid Tests ...........................................................92
Fluorescence Liquid Tests..........................................................99
Luminescence Test...................................................................119
Dispense Module Tests .............................................................124
80 | Chapter 5: Instrument Qualification
Overview
This chapter contains BioTek Instruments’ recommended Installation Qualification (IQ),
Operational Qualification (OQ), and Performance Qualification (PQ) procedures for the
Synergy 2.
Every Synergy 2 reader and external dispense module is fully tested at BioTek prior to
shipment and should operate properly upon initial setup. If you suspect that a problem
occurred during shipment, if you have received the equipment after returning it to the
factory for service, and/or if regulatory requirements dictate that you qualify the
equipment on a routine basis, you should perform the procedures outlined in this chapter.
™ A Product Qualification Package (PN 7130566) for the Synergy 2 is
available for purchase. The package contains complete procedures for
performing Installation Qualification, Operational Qualification,
Performance Qualification, and Preventive Maintenance. Gen5
protocols with embedded Microsoft® Excel spreadsheets are provided
for performing the tests and analyzing results. Checklists, data sheets,
and logbooks are also provided. Contact your local BioTek dealer for
more information.
IQ/OQ/PQ
Installation Qualification confirms that the reader and its components have been
supplied as ordered and ensures that they are assembled and configured properly for your
lab environment.
•
The recommended IQ procedure consists of setting up the instrument and its
components as described in Chapter 2, Installation, and performing the System
Test. For models with injectors, a quick “Injector Test” is also performed, to ensure
that the dispense module is properly installed and there are no leaks.
•
The IQ procedure should be performed initially (before the reader is used for the
first time).
•
The successful completion of the IQ procedure verifies that the instrument is
installed correctly. The Operational Qualification procedure should be performed
immediately following the successful IQ.
BioTek Instruments, Inc.
IQ/OQ/PQ |
81
Operational Qualification confirms that the equipment operates according to specification
initially and over time.
•
The recommended OQ procedure consists of performing the System Test,
Absorbance Plate Test, a series of Liquid Tests, and, if the external dispense
module is used, the Dispense Accuracy and Precision Tests.
•
Your facility’s operating policies may also require that you perform an actual assay
prior to accepting the reader for routine use. If this is the case, you should not use
the data obtained from the first assay run on the reader until you have confirmed
that the package insert criteria have been met.
•
The OQ procedure should be performed initially (before first use) and then
routinely; the recommended interval is annually. It should also be performed after
any major repair or upgrade to the hardware or software.
•
Although out-of-tolerance failures will be detected by the OQ tests, results should
be compared with those from the routine Performance Qualification tests and
previous OQ tests to monitor for trends.
•
The successful completion of the OQ procedure, in combination with results that
are comparable to previous PQ and OQ tests, confirms that the equipment is
operating according to specification initially and over time.
Performance Qualification confirms that the reader consistently meets the requirements
of the tests performed at your laboratory.
•
The recommended PQ procedure consists of performing the System Test,
Absorbance Plate Test, a series of Liquid Tests, and, if the external dispense
module is used, the Dispense Accuracy and Precision Tests.
•
Your facility’s operating policies may also require that you routinely perform an
actual assay, to confirm that the reader will consistently give adequate results for
the assays to be run on it.
•
These tests should be performed routinely; the recommended interval is monthly
or quarterly, depending on the test. This frequency may be adjusted depending on
the trends observed over time.
•
The successful completion of the PQ procedure confirms that the equipment is
performing consistently under normal operating conditions.
Synergy 2 Operator’s Manual
82 | Chapter 5: Instrument Qualification
Recommended Qualification Schedule
The schedule below defines the factory-recommended intervals for qualifying a Synergy 2
used two to five days a week. The actual frequency, however, may be adjusted depending
on your usage of the instrument and its various modules. This schedule assumes the
reader is properly maintained as outlined in Chapter 6, Preventive Maintenance.
IQ
OQ
Initially
Initially/
Annually
Monthly
9
9
Absorbance Plate Test
9
9
Absorbance Liquid Test 1 or 2*
9
9
Absorbance Liquid Test 3 (optional)**
9
9
Tasks/Tests
PQ
Quarterly
All models:
Installation, setup, and configuration of the
reader, dispense module (if equipped), the
host computer, and Gen5 software
9
System Test
9
Models with the Absorbance module:
Models with the Fluorescence module(s):
Corners, Sensitivity, Linearity (FI) Tests
9
Fluorescence Polarization (FP) Tests***
9
9
Time-Resolved Fluorescence (TRF) Test***
9
9
9
Models with the Luminescence module:
Luminescence Test
9
9
Models with the Dispense module:
Injection System Test
Dispense Accuracy and Precision Test
9
9
9
* Regarding Absorbance Liquid Tests 1 and 2:
•
If you have an Absorbance Test Plate, run Liquid Test 1.
•
If you do not have an Absorbance Test Plate, run Liquid Test 2.
** Liquid Test 3 is optional; it is provided for sites requiring verification at wavelengths
lower than those attainable with the Absorbance Test Plate.
*** If applicable to your reader model.
BioTek Instruments, Inc.
System Test |
83
System Test
Each time the Synergy 2 is turned on, it automatically performs a series of tests on the
reader’s motors, lamps, the PMT, and various sub-systems. The duration of this “System
Test” depends on the reader model, and can a few minutes to complete. If all tests pass, the
microplate carrier will eject and the LED on the power switch will remain on and constant.
The reader is then ready for use.
If any test results do not meet the internally-coded Failure Mode Effects Analysis (FMEA)
criteria established by BioTek, the reader will beep repeatedly and the LED on the power
switch will flash repeatedly. If this occurs, press the Carrier Eject button to stop the
beeping. If necessary, initiate another System Test using Gen5 to try to retrieve an error
code from the reader. Refer to Appendix B, Error Codes for information on error codes
and for troubleshooting tips.
To run the System Test:
1. Turn on the reader and launch Gen5.
2. If applicable, set Gen5’s Absorbance wavelength table to the six wavelengths you
most frequently use.
3. If your assays use incubation, we recommend enabling Temperature Control and
allowing the incubator to reach its set point before running the System Test. To
access this feature, select System > Reader Control and click the Pre-Heating tab.
4. Select System > Diagnostics > Run System Test.
™ If the test fails during execution, a message box will appear in the
software. Close the box; the test report will contain the error code that
was generated by the failure.
5. When the test is complete, a dialog will appear, requesting additional information.
Enter your User name and other information (if desired) and then click OK.
6. The test report will appear. Scroll down toward the bottom of the report; it will show
either “SYSTEM TEST PASS” or “SYSTEM TEST FAIL *** ERROR (error code)
DETECTED.”
7. Print the report if desired.
•
A sample test report is shown on the next few pages.
•
Gen5 stores the results in a database, so the results can be retrieved at any time.
We recommend that you print and save the reports to document that the test
was performed.
8. If the test failed, look up the error code in Appendix B, Error Codes to determine its
cause. If the cause is something you can fix, turn off the reader, fix the problem, and
then turn the reader back on and retry the test. If the test continues to fail, or if the
cause is not something you can fix, contact BioTek’s Technical Assistance Center.
Synergy 2 Operator’s Manual
84 | Chapter 5: Instrument Qualification
™ A sample test report for a Synergy 2 with all modules installed is
shown on the next few pages. Your instrument’s test report may be
different. If you have any questions about the report’s contents or the
test results, please print the report and then contact BioTek’s
Technical Assistance Center.
Gen5 System Test Report
Reader:
Basecode:
Date and Time:
User:
Company:
Comments:
Synergy 2 (Serial Number: 200351)
P/N 7130202 (v1.02)
6/22/2009 3:28:35 PM
BioTek User
Test Results
Operator ID:_________________________________________________________
Notes:_______________________________________________________________
_____________________________________________________________________
SYSTEM SELF TEST
7130202
Version 1.02
Bias current offset
Offset voltage
750V measurement
750V noise
500V measurement
500V noise
Reference bias
Reference offset
Reference noise
Excitation Wheel
Filter 1:
Gain:
Air:
Dark:
Delta:
Filter 2:
Gain:
Air:
Dark:
Delta:
Filter 3:
Gain:
Air:
Dark:
Delta:
Filter 4:
360/40
1.00
15503
10982
4521
485/20
1.00
15697
10982
4715
540/25
1.00
15226
10982
4244
PLUG
Emission Wheel
Filter 1:
Filter 2:
460/40
528/20
200351
-2.2
1501
24.3
15
6.4
1
0.7
10982
0.2
0111 1111 0000 0000
counts
counts
counts
counts
counts
counts
counts
counts
counts
1.00
17693
10982
6711
1.00
18034
10982
7052
1.00
18706
10982
7724
1.00
17195
10983
6212
1.00
17672
10982
6690
1.00
18043
10983
7060
1.00
16596
10982
5614
1.00
16866
10983
5883
1.00
17135
10982
6153
PASS
PASS
PASS
BioTek Instruments, Inc.
System Test |
Filter 3:
Filter 4:
620/40
OPEN
Absorbance
Wavelength 1:
Channel:
Air:
Dark:
Delta:
Wavelength 2:
Channel:
Air:
Dark:
Delta:
Wavelength 3:
Channel:
Air:
Dark:
Delta:
Wavelength 4:
Channel:
Air:
Dark:
Delta:
Wavelength 5:
Channel:
Air:
Dark:
Delta:
Wavelength 6:
Channel:
Air:
Dark:
Delta:
260
Ref
13096
9949
3147
280
Ref
12950
9958
2992
405
Ref
12602
9965
2637
450
Ref
12537
9956
2581
490
Ref
12487
9957
2530
630
Ref
12373
9978
2395
Gain:
Meas
39826
9947
29879
Gain:
Meas
39368
9954
29414
Gain:
Meas
39343
9960
29383
Gain:
Meas
39774
9952
29822
Gain:
Meas
39975
9953
30022
Gain:
Meas
39621
9968
29653
Noise Test
Channel:
Noise Max:
Noise Min:
Delta:
Ref
10046
10045
1
Meas
10028
10027
1
Voltage Reference:
Lamp:
Motor:
Absorbance Flash:
Fluorescence Flash:
Min
3375
3723
1314
664
1.41
Resets: 8
1.87
Resets: 4
2.82
Resets: 8
1.66
Resets: 4
2.07
Resets: 8
2.51
Resets: 2
Low
High
Max
1596
834
1982
878
2264
915
INCUBATOR SELF TEST
Temperature Setpoint:
Zone
Zone
Zone
Zone
1:
2:
3:
4:
37.3
37.1
37.2
37.3
Min:
Min:
Min:
Min:
37.0
37.2
37.0
37.1
37.2
Current Average: 37.2
Max:
Max:
Max:
Max:
37.4
37.2
37.3
37.4
AUTOCAL ANALYSIS
PROBE:
BOTTOM
Upper Left Corner: x=
Lower Left Corner: x=
Lower Right Corner: x=
9708
9696
1016
Synergy 2 Operator’s Manual
y= 4536
y=10068
y=10072
Range:
Range:
Range:
Range:
PASS
PASS
PASS
PASS
A/D Test: PASS
Thermistor:
Thermistor:
Thermistor:
Thermistor:
PASS
PASS
PASS
PASS
85
86 | Chapter 5: Instrument Qualification
Upper
Delta
Delta
Delta
Delta
Right Corner: x=
1: 9708 - 9696=
2: 1024 - 1016=
3: 4540 - 4536=
4: 10072 -10068=
1024
+12
+8
+4
+4
y= 4540
Left Corner: x= 10608
Left Corner: x= 10596
Right Corner: x= 1916
Right Corner: x= 1920
1: 10608 -10596=
+12
2: 1920 - 1916=
+4
3:
196 - 192=
+4
4: 5724 - 5724=
+0
y= 192
y= 5724
y= 5724
y= 196
PROBE:
Upper
Lower
Lower
Upper
Delta
Delta
Delta
Delta
PROBE:
Upper
Lower
Lower
Upper
Delta
Delta
Delta
Delta
LUMIN
Left Corner: x=
Left Corner: x=
Right Corner: x=
Right Corner: x=
1: 9696 - 9692=
2: 1012 - 1008=
3: 2984 - 2988=
4: 8516 - 8516=
PROBE:
Upper
Lower
Lower
Upper
Delta
Delta
Delta
Delta
TOP
9696
9692
1008
1012
+4
+4
-4
+0
y=
y=
y=
y=
2988
8516
8516
2984
ABSORB
Left Corner: x= 11252
Left Corner: x= 11240
Right Corner: x= 2556
Right Corner: x= 2564
1: 11252 -11240=
+12
2: 2564 - 2556=
+8
3: 4544 - 4536=
+8
4: 10068 -10064=
+4
y= 4536
y=10064
y=10068
y= 4544
Monochromator:
A=+0.000000
Probe Height:
Plate Sensor:
33.93 mm
180
Probe Changer:
572
Mirror:
3864
Lamp Reflector:
1024
Aperture:
2356
Middle Sensor:
Tested:
Delta:
y= 11964
11972
+8
Back Sensor:
Tested:
Delta:
x= 11572
11568
-4
B=-0.001461
C=-0.147723
y=10688
10688
+0
SYSTEM TEST PASS
0000
BioTek Instruments, Inc.
Absorbance Plate Test |
87
Dispenser 1: 005.1,010.0,020.0,040.0,080.0,200.1
Dispenser 2: 005.0,010.0,020.0,040.0,080.0,200.2
Mirror 1: 50% Mirror, 0200, 0850, 0200, 0850
Mirror 2: Dichroic , 0320, 0400, 0420, 0800
Mirror 3: Dichroic , 0440, 0500, 0510, 0780
Polarizers present.
Reviewed/Approved By: _______________________________________
Date: ________________
Figure 37: Sample output for the System Test
Absorbance Plate Test
Description
This test uses BioTek’s Absorbance Test Plate (PN 7260522) to confirm mechanical
alignment; optical density accuracy, linearity, and repeatability; and wavelength
accuracy. The Absorbance Plate Test compares the reader’s optical density and
wavelength measurements to NIST-traceable values.
•
Mechanical Alignment: The Test Plate has precisely machined holes to confirm
the mechanical alignment of the reader. The amount of light that shines
through these holes is an indication of whether the reader is properly aligned
with the absorbance optical path. A reading of more than 0.015 OD for any of
the designated alignment holes indicates that the light is being “clipped” and
the reader may be out of alignment.
•
Accuracy/Linearity: The Test Plate contains neutral-density glass filters of
known OD values at several wavelengths. Actual measurements are compared
against the expected values provided in the Test Plate’s Standards Certificate.
Since there are several filters with differing OD values, the accuracy across a
range of ODs can be established. Once it is proven that the reader is accurate at
these OD values, the reader is also considered to be linear.
•
Repeatability: This test ensures the instrument meets its repeatability
specification by reading each neutral-density filter on the Test Plate twice with
the filter in the same location.
•
Wavelength Accuracy: The Test Plate contains a glass filter in position C6; it is
used to check the wavelength accuracy of the absorbance monochromator. The
filter is scanned across a specified wavelength range in 1-nm increments. The
wavelength of maximum absorbance is compared to the expected peak
wavelength supplied on the Test Plate’s certificate.
™ An alternate method for determining accuracy, linearity, and
repeatability is Absorbance Liquid Test 2, described on page 95.
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88 | Chapter 5: Instrument Qualification
Test Plate Certificates
To run this test on the Synergy 2, you’ll need BioTek’s 7-Filter Absorbance Test Plate
(PN 7260522), with its accompanying certificates.
•
The Standards Certificate contains standard OD values for the filters at several
different wavelengths (see the sample below).
•
The Peak Wavelength Certificate contains one or more “Peak Wavelength”
values for the glass filter in position C6 on the plate. Each value has a valid test
range associated with it. For example, a Peak Wavelength value may be 586 nm
with a test range of 580 to 590 nm (or tolerance values of -6/+4).
This test plate can be used for testing the reproducibility, linearity, and
alignment of your BioTek autoreader. The following calibration data has
been recorded by a N.I.S.T. traceable spectrophotometer.
WAVELENGTH (nm)
Well
405nm
450nm
490nm
550nm
620nm
630nm
690nm
750nm
C1
0.147
0.140
0.135
0.130
0.136
0.136
0.127
0.134
E2
0.618
0.575
0.574
0.568
0.573
0.568
0.485
0.434
G3
1.133
1.052
1.051
1.040
SAM
P L1.050
E
1.040
0.881
0.783
H6
1.701
1.578
1.577
1.560
1.575
1.560
1.323
1.179
F5
2.279
2.024
1.976
1.956
1.893
1.865
1.537
1.272
D4
2.945
2.604
2.545
2.513
2.437
2.400
1.972
1.632
Set # 2453
Serial # 161259
Figure 38: Sample Standards Certificate with OD/Wavelength combinations
for each of six locations in the Absorbance Test Plate
Before the Absorbance Plate Test can be performed, the standard OD values and the
peak wavelength value(s) must be entered into Gen5. You’ll enter and save these
values once initially, and then update them each time the test plate is recertified by
BioTek (typically annually).
Define Absorbance Test Plate Parameters
™ Gen5 version 1.07 and earlier users only: The Gen5 Reader
Diagnostics Utility must be installed to run the Absorbance Plate Test.
1. Obtain the certificates that came with the Test Plate.
2. Start Gen5 and select System > Diagnostics > Test Plates > Add/Modify
Plates.
3. Click Add. The Absorbance Test Plate dialog will appear.
4. Select the appropriate Plate Type and enter the plate’s Serial Number.
BioTek Instruments, Inc.
Absorbance Plate Test |
89
5. Enter the Last Certification and Next Certification dates from the calibration sticker
on the Test Plate.
6. If the wavelength values in the top row of the grid are appropriate for your tests,
enter the OD values from the Standards Certificate into the grid. Make sure you
enter the correct value for each well/wavelength combination.
•
If you need to change the wavelength values, click Wavelength List. Click the
Gen5 Help button for assistance.
7. Select the number of Peak Wavelength tests to run (1 to 4), based on the number of
peak wavelength values provided on the Peak Wavelength Certificate.
8. Enter the Expected Peak value(s) from the Peak Wavelength Certificate. (If multiple
values are given for a wavelength, use those in the 2.4 nm Spectral Bandpass table.)
For each value, define the expected Test Range by selecting the minus/plus tolerance
values. The range must span at least 8 nm.
™ For certificates that have only one peak wavelength and a fixed
wavelength range of 580 to 590 nm, enter the Expected Peak
wavelength value and adjust the Test Range values so the range
displayed in parentheses is 580 to 590 (as demonstrated above).
9. Review all of the values you entered, and then click OK to save the data.
The information you just entered will be available in Gen5 each time the Absorbance
Plate Test is performed. It may need to be modified after the annual recertification of
your test plate.
Run the Absorbance Plate Test
1. In Gen5, select System > Diagnostics > Test Plates > Run.
2. If prompted, select the desired Test Plate and click OK.
3. When the Absorbance Test Plate Options dialog appears, select Perform Peak
Wavelength Test if it is not already selected.
4. Highlight the wavelength(s) to be included in this test. You only need to select those
wavelengths most appropriate for your use of the reader.
5. (Optional) Enter any Comments.
6. Click Start Test.
7. Place the Test Plate in the microplate carrier so that well A1 is in the left-rear corner
of the carrier.
8. Click OK to run the test.
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90 | Chapter 5: Instrument Qualification
9. When the test is completed, the results report appears. Scroll through the report;
every result should show “PASS”. See page 91 for information on results and
troubleshooting tips in the event of failures.
•
A sample test report is shown below.
•
Gen5 stores the results in a database; they can be retrieved any time. We
recommend you print and save the report to document that the test was
performed.
Sample Report
Absorbance Test Plate Results
Reader:
Basecode:
Date and Time:
Absorbance Plate:
Last Plate Certification:
Next Plate Certification Due:
User:
Comments:
Synergy 2 (Serial Number: 200363)
P/N 7130202 (v1.02)
11/16/2009 10:01:05 AM
7 Filter Test Plate (P/N 7260522) - S/N 179269
September 2009
September 2010
BioTek User
Test run during initial OQ
Peak Absorbance Results
Well
Reference
Tolerance
Read
Result
C6
586
3
586
PASS
Alignment Results
Wells
A1
Read
0.002
Tolerance
0.015
Result
PASS
Wavelength = 405 nm
A12
0.002
0.015
PASS
H1
0.003
0.015
PASS
H12
0.002
0.015
PASS
Accuracy Results
Wells
Reference
Min Limit
Max Limit
Read 1
Result
C1
0.140
0.117
0.163
0.144
PASS
E2
0.632
0.599
0.665
0.633
PASS
G3
1.200
1.156
1.244
1.201
PASS
H6
1.744
1.689
1.799
1.744
PASS
F5
2.076
1.973
2.179
2.074
PASS
D4
2.679
2.552
2.806
2.691
PASS
G3
1.201
1.184
1.218
1.201
PASS
H6
1.744
1.722
1.766
1.745
PASS
F5
2.074
2.007
2.141
2.074
PASS
D4
2.691
2.605
2.777
2.688
PASS
Repeatability Results
Wells
Read 1
Min Limit
Max Limit
Read 2
Result
C1
0.144
0.138
0.150
0.144
PASS
E2
0.633
0.622
0.644
0.633
PASS
Figure 39: Sample Absorbance Plate Test Report
BioTek Instruments, Inc.
Absorbance Plate Test |
91
Results & Troubleshooting Tips
The Absorbance Test Plate Report contains results for the following:
•
•
•
Peak Absorbance: When the test is performed, the C6 filter is scanned at the
test range(s) defined by the user in the Absorbance Test Plate dialog. To verify
wavelength accuracy, the wavelength of the maximum absorbance is compared
with the peak wavelength value entered in the software, which comes from the
Peak Wavelength Certificate supplied with the Test Plate. The accuracy of the
wavelength should be ± 3 nm (± 2 nm instrument, ± 1 nm filter allowance). If
the reader fails this test:
¾
Make sure the information entered into Gen5 matches the information on
the Test Plate’s Peak Wavelength Certificate.
¾
Verify that the Test Plate has a filter in location C6. (Test Plates with part
number 9000547 do not have this filter.)
¾
Check the C6 filter to make sure it is clean. If needed, clean it with lens
paper. Do not remove the filter from the Test Plate, and do not use
alcohol or other cleaning agents.
¾
Make sure the Test Plate is within its calibration certification period. If it
is out of date, contact BioTek to schedule a recertification.
¾
Check the microplate carrier to ensure it is clear of debris.
Alignment: This portion of the test measures the alignment of the microplate
carrier with the optical path. A reading greater than 0.015 OD represents an
out-of-alignment condition. If the reader fails this test:
¾
Ensure that the Test Plate is correctly seated in the microplate carrier.
¾
Check the four alignment holes (A1, A12, H1, H12) to ensure they are
clear of debris.
¾
Check the microplate carrier to ensure it is clear of debris.
Accuracy: Accuracy is a measure of the optical density of Test Plate wells C1,
D4, E2, F5, G3, and H6 as compared with known standard values contained in
the test plate’s Standards Certificate. If the reader fails this test:
¾
Verify that the filter calibration values entered in Gen5 are the same as
those on the Test Plate’s Standards Certificate.
¾
Check the neutral-density filters on the Test Plate to ensure they are
clean. If needed, clean them with lens paper. Do not remove the filters
from the test plate, and do not use alcohol or other cleaning agents.
¾
Make sure the Test Plate is within its calibration certification period. If it
is out of date, contact BioTek to schedule a recertification.
Linearity of the optical density readings is confirmed by default if the optical
density readings are accurate. To further verify this, you can perform a
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92 | Chapter 5: Instrument Qualification
regression analysis on the Test Plate OD values in a spreadsheet program such
as Microsoft Excel. An R Square value of at least 0.990 is expected.
•
Repeatability: Repeatability is a measure of the instrument’s ability to read the
same well with minimum variation between two reads with the well in the
same location. If the reader fails this test:
¾
Check the filters on the Test Plate to ensure there is no debris that may
have shifted between readings and caused changes.
¾
Check the microplate carrier to ensure it is clear of debris.
Absorbance Liquid Tests
Conducting Liquid Tests confirms the reader’s ability to perform to specification with
liquid samples. Liquid testing differs from testing with the Absorbance Test Plate in that
liquid in the wells has a meniscus, whereas the Test Plate’s neutral density glass filters do
not. The optics characteristics may differ in these two cases, thus alerting the operator to
different types of problems.
•
Liquid Test 1 confirms repeatability and alignment of the reader when a solution
is used in the microplate. If these tests pass, then the lens placement and optical
system cleanliness are proven.
•
The recommended method for testing the instrument’s alignment, repeatability,
and accuracy is to use the Absorbance Test Plate (see page 87). If the Test Plate is
not available, however, Liquid Test 2 can be used for these tests.
•
Liquid Test 3 verifies operation of the reader at 340 nm, and is provided for sites
requiring proof of linearity at wavelengths lower than those attainable with the
Absorbance Test Plate. This test is optional because the reader has good “front
end” linearity throughout its wavelength range.
BioTek Instruments, Inc.
Absorbance Liquid Tests |
93
Absorbance Liquid Test 1
Materials
™ Manufacturer part numbers are subject to change.
•
New 96-well, clear, flat-bottom microplate (Corning Costar #3590
recommended)
•
Stock Solution A or B, which may be formulated by diluting a dye solution
available from BioTek (A) or from the ingredients listed below (B).
Solution A
•
BioTek QC Check Solution No. 1 (PN 7120779, 25 mL; PN 7120782, 125 mL)
•
Deionized water
•
5-mL Class A volumetric pipette
•
100-mL volumetric flask
1. Pipette a 5-mL aliquot of BioTek QC Check Solution No. 1 into a 100-mL
volumetric flask.
2. Add 95 mL of DI water; cap and shake well. The solution should measure
approximately 2.000 OD when using 200 µL in a flat-bottom microwell.
Solution B
•
Deionized water
•
FD&C Yellow No. 5 dye powder (typically 90% pure)
•
Tween 20 (polyoxyethylene (20) sorbitan monolaurate) or BioTek
wetting agent (PN 7773002) (a 10% Tween solution)
•
Precision balance with capacity of 100 g minimum and readability of 0.001 g
•
Weigh boat
•
1-liter volumetric flask
1. Weigh out 0.092 g of FD&C Yellow No. 5 dye powder into a weigh boat.
2. Rinse the contents into a 1-liter volumetric flask.
3. Add 0.5 mL of Tween 20, or 5 mL of BioTek’s wetting agent.
4. Fill to 1 liter with DI water; cap and shake well. The solution should measure
approximately 2.000 OD when using 200 µL in a flat-bottom microwell.
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94 | Chapter 5: Instrument Qualification
Prepare the Plate
™ Use a new microplate. Fingerprints or scratches may cause variations
in readings.
1. Using freshly prepared stock solution (Solution A or B), prepare a 1:2 dilution
using deionized water (one part stock, one part deionized water; the resulting
solution is a 1:2 dilution).
2. Pipette 200 µL of the concentrated solution (A or B) into the first column of wells
in the microplate.
3. Pipette 200 µL of the diluted solution into the second column of wells.
™ After pipetting the diluted test solution into the microplate and before
reading the plate, we strongly recommend shaking the plate at
Variable speed for four minutes. This will allow any air bubbles in the
solution to settle and the meniscus to stabilize. Alternatively, wait 20
minutes after pipetting the test solution before reading the plate.
Read the Plate
1. Using Gen5, read the microplate five times at 405 nm using the Normal read
mode, single wavelength, no blanking. Save the data after each read (“Normal”
plate position).
2. Without delay, rotate the microplate 180 degrees so that well A1 is in the “H12”
position. Read the plate five more times, saving the data after each read
(“Turnaround” plate position).
3. Print out the ten sets of raw data, or export them to an Excel spreadsheet.
Analyze the Results
1. The plate is read five times in the “Normal” position at 405 nm. Calculate the
Mean OD and Standard Deviation of those five reads for each well in columns 1
and 2.
2. For each well in columns 1 and 2, calculate the Allowed Deviation using the
repeatability specification for a 96-well plate: ± 1% ± 0.005 OD (Mean x 0.010 +
0.005). For each well, its standard deviation should be less than its allowed
deviation.
Example: Five readings in well A1 of 0.802, 0.802, 0.799, 0.798, and 0.801 result in
a mean of 0.8004 and a standard deviation of 0.0018. The mean multiplied by
1.0% (0.8004 * 0.010) equals 0.008, and when added to 0.005 equals 0.013; this is
the allowed deviation for well A1. Since the standard deviation for well A1 is less
than 0.013, the well meets the test criteria.
3. The plate is read five times in the “Turnaround” position at 405 nm. Calculate
the Mean OD of those five reads for each well in columns 11 and 12.
BioTek Instruments, Inc.
Absorbance Liquid Tests |
95
4. Perform a mathematical comparison of the Mean values for each microwell in its
Normal and Turnaround positions (that is, compare A1 to H12, A2 to H11, B1 to
G12,… H2 to A11). To pass the test, the differences in the compared mean values
must be within the accuracy specification for a 96-well microplate: ± 1.0% ± 0.010
OD from 0.000 to 2.000 OD.
Example: If the mean value for well A1 in the Normal position is 1.902 with a
specified accuracy of ± 1.0% ± 0.010 OD, then the expected range for the mean of
the well in its Turnaround (H12) position is 1.873 to 1.931 OD. 1.902 x 0.010 +
0.010 = 0.029; 1.902 - 0.029 = 1.873; 1.902 + 0.029 = 1.931.
Absorbance Liquid Test 2
Materials
•
A new 96-well, clear, flat-bottom microplate (Corning Costar #3590 is
recommended)
•
Ten test tubes, numbered consecutively, set up in a rack
•
Calibrated hand pipette (Class A volumetric pipette recommended)
•
Solution A or B (see the instructions on page 93)
•
A 0.05% solution of deionized water and Tween 20
Prepare the Dilutions
Create a percentage dilution series, beginning with 100% of the original
concentrated stock solution (A or B) in the first tube, 90% of the original solution in
the second tube, 80% in the third tube, all the way to 10% in the tenth tube. Dilute
using the 0.05% solution of deionized water and Tween 20. This solution can also
be made by diluting the BioTek wetting agent 200:1.
Tube Number:
Volume of Original
Concentrated Solution (mL)
Volume of 0.05% Tween
Solution (mL)
Absorbance expected if original
solution is 2.0 at 200 μL
1
2
3
4
5
6
7
8
9
20
18
16
14
12
10
8
6
4
2
0
2
4
6
8
10
12
14
16
18
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
™ The choice of dilutions and the absorbance of the original solution can
be varied. Use this table as a model for calculating the expected
absorbances of a series of dilutions, given a different absorbance of
the original solution.
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96 | Chapter 5: Instrument Qualification
Prepare the Plate
•
Pipette 200 µL of the concentrated solution from Tube 1 into each well of the
first column, A1 to H1, of a new flat-bottom microplate.
•
Pipette 200 µL from each of the remaining tubes into the wells of the
corresponding column of the microplate (Tube 2 into wells A2 to H2, Tube 3
into wells A3 to H3, and so on).
Linearity & Repeatability Tests
1. Using Gen5, read the microplate prepared above five times using Normal mode,
dual wavelength at 450/630 nm. Save the data after each read.
™ Do not discard the plate; you will use it for the Alignment test.
2. Print out the five sets of Delta OD data, or export them to an Excel spreadsheet.
3. Calculate the results for Linearity:
•
Calculate the mean absorbance for each well, and average the means for
each concentration.
•
Perform a regression analysis on the data to determine if there is adequate
linearity. Since it is somewhat difficult to achieve high pipetting accuracy
when conducting linear dilutions, an R Square value of at least 0.99 is
considered adequate.
4. Calculate the results for Repeatability:
•
Calculate the mean and standard deviation for the five readings taken in
Step 1 at each concentration. Only one row of data needs to be analyzed.
•
For each mean below 2.000 OD, calculate the allowed deviation using the
repeatability specification for a 96-well plate of ± 1.0% ± 0.005 OD. (If above
2.000 OD, apply the ± 3.0% ± 0.005 specification.)
•
The standard deviation for each set of readings should be less than the
allowed deviation.
Example: Absorbance readings of 1.950, 1.948, 1.955, 1.952, and 1.950 result
in a mean of 1.951, and a standard deviation of 0.0026. The mean (1.951)
multiplied by 1.0% (1.951 x 0.010) = 0.0195, which, when added to the 0.005
(0.0195 + 0.005) = 0.0245 OD, which is the allowable deviation. Since the
standard deviation is less than this value, the reader meets the test criteria.
Alignment Test
1. Using the plate prepared for the Linearity Test, conduct a Turnaround test by
reading the plate five times with the A1 well in the H12 position. Save the data
after each read.
BioTek Instruments, Inc.
Absorbance Liquid Tests |
97
2. Calculate the means of the wells A1 and H1 in the Normal plate position (data
from Linearity Test) and in the Turnaround position (from Step 1).
3. Compare the mean reading for well A1 to its mean reading when in the H12
position. Next, compare the mean values for the H1 well to the same well in the
A12 position. The difference in the values for any two corresponding wells
should be within the accuracy specification for a 96-well plate, ± 1.0% ± 0.010 OD
from 0.000 to 2.000 OD.
Example: If the mean of well A1 in the normal position is 1.902, where the
specified accuracy is ± 1.0% ± 0.010 OD, then the expected range for the mean of
the same well in the H12 position is 1.873 to 1.931 OD. (1.902 x 1.0% = 0.019 +
0.010 = 0.029, which is added to and subtracted from 1.902 for the range.)
If the four corner wells are within the accuracy range, the reader is in alignment.
Absorbance Liquid Test 3 (Optional)
Materials
™ Manufacturer part numbers are subject to change.
•
New 96-well, clear, flat-bottom microplate (Corning Costar #3590
recommended)
•
Calibrated hand pipette(s)
•
Beakers and graduated cylinder
•
Precision balance with readability to 0.01 g
•
Buffer solution described below
Buffer Solution
•
Deionized water
•
Phosphate-Buffered Saline (PBS), pH 7.2–7.6, Sigma tablets, #P4417 (or
equivalent)
•
β-NADH Powder (β-Nicotinamide Adenine Dinucleotide, Reduced Form)
Sigma bulk catalog number N 8129, or preweighed 10-mg vials, Sigma
number N6785-10VL (or BioTek PN 98233). Store the powder according to
the guidelines on its packaging.
1. Prepare a PBS solution from the Sigma tablets.
2. In a beaker, mix 50 mL of the PBS solution with 10 mg of the β-NADH
powder and mix thoroughly. This is the 100% Test Solution.
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98 | Chapter 5: Instrument Qualification
3. (Optional) Read a 150-µL sample of the solution at 340 nm; it should be
within 0.700 to 1.000 OD. If low, adjust up by adding more powder. Do not
adjust if slightly high.
Prepare the Plate
1. Prepare the 75% Test Solution by mixing 15 mL of the 100% Test Solution with
5 mL of the PBS Solution.
2. Prepare the 50% Test Solution by mixing 10 mL of the 100% Test Solution with
10 mL of the PBS Solution.
3. Carefully pipette the three solutions into a new 96-well microplate:
•
150 µL of the 100% Test Solution into all wells of columns 1 and 2
•
150 µL of the 75% Test Solution into all wells of columns 3 and 4
•
150 µL of the 50% Test Solution into all wells of column 5 and 6
Read the Plate
1. Using Gen5, read the microplate five times using Normal mode, single
wavelength at 340 nm, no blanking. Save the data after each read.
2. Print out the five sets of raw data, or export them to an Excel spreadsheet.
Analyze the Results
1. For each well, calculate the Mean OD and Standard Deviation of the five
readings.
2. For each mean calculated in step 1, calculate the allowed deviation using the
repeatability specification for a 96-well plate: ± 1% ± 0.005 OD (Mean x 0.010 +
0.005). For each well, its standard deviation should be less than its allowed
deviation.
Example: Five readings in well A1 of 0.802, 0.802, 0.799, 0.798, and 0.801 result in
a mean of 0.8004 and a standard deviation of 0.0018. The mean multiplied by
1.0% (0.8004 * 0.010) equals 0.008, and when added to 0.005 equals 0.013; this is
the allowed deviation for well A1. Since the standard deviation for well A1 is less
than 0.013, the well meets the test criteria.
3. Calculate the results for Linearity:
•
For each of the three Test Solutions, calculate the average Mean OD for the
wells containing that solution (mean of wells A1-H2, A3-H4, and A5-H6).
•
Perform a regression analysis on the data to determine if there is adequate
linearity. The three average Mean OD values are the “Y” values. The
solution concentrations are the “X” values (1.00, 0.75, 0.50). Since it is
somewhat difficult to achieve high pipetting accuracy when conducting
linear dilutions, an R Square value of at least 0.99 is considered adequate.
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
99
Fluorescence Liquid Tests
For Synergy 2 models with fluorescence capability, BioTek has developed a series of
module-specific liquid tests for verifying performance.
•
Corners test: Verifies that the plate carrier is properly aligned in relation to the
fluorescence probes.
•
Sensitivity test: Verifies the fluorescence reading capability of the reader. The
ability to detect specific compounds at low concentrations ensures that the filters,
optical path, and PMT are all in working order. This test verifies that the difference
between the mean of wells with known lower limits of concentration of the
substance under investigation is statistically distinguishable from the mean of
wells with pure diluent.
•
Linearity test: Verifies that the system is linear; that is, signal changes
proportionally with changes in concentration. Proving that the system is linear
allows the Sensitivity Test to be run on two points instead of using serial dilutions.
•
FP test: Verifies the ability of the instrument to measure polarization of the
solution properly. It verifies the polarizers are installed in the proper orientation,
and the mechanism is in proper order.
•
TRF test: Verifies the performance of the xenon flash bulb and that the filters,
optical path, and PMT are all in working order.
The tests presented in this section require specific microplates, solutions,
filters, and mirrors. Your laboratory may require a deviation from some
of these tests. For example, you may wish to use a different fluorescing
solution, dichroic mirror, and/or microplate.
If deviation from the tests as presented in this section is required, the
following steps should be taken the first time each test is run (e.g.,
during the Initial OQ):
1 Perform the tests exactly as described on the following pages.
2 Re-run the tests using your particular solutions, filters, mirrors,
microplates, etc. If results are comparable, then the results from these
tests will be your baseline for future tests.
3 Be sure to document your new test procedure(s), and save all test
results.
Synergy 2 Operator’s Manual
100 | Chapter 5: Instrument Qualification
Required Materials
™ BioTek offers test kits containing the microplates and solutions used
in fluorescence liquid test procedures; see page 23.
™ Microplates should be perfectly clean and free of dust or bottom
scratches. Use new microplates from sealed packages.
All Tests
•
Deionized or distilled water
•
Various beakers, graduated cylinders, and pipettes
•
95% ethanol (for cleaning clear-bottom plates)
•
Aluminum foil
•
(Optional, but recommended) 0.45-micron filter
•
(Optional) Black polyethylene bag(s) to temporarily store plate(s)
•
The “Fluorescence Liquid Tests” Gen5 protocols described starting on page 111
Corners/Sensitivity/Linearity (FI) Tests
™ Methylumbelliferone can be used as an alternative or supplemental
method for performing these tests for the top probe. See page 114.
•
Bottom optics: A clean Hellma® Quartz 96-well titration plate (Mfr.
#730.009.QG), or equivalent, such as the 96-well glass-bottom Greiner
SensoPlate™ (Mfr. #655892).
•
Top optics: A new, clean, 96-well solid black microplate, such as Corning®
Costar Mfr. #3915. The Greiner SensoPlate mentioned above can also be used.
•
Buffer:
¾ NIST-traceable Sodium Borate Reference Standard (pH 9.18), (e.g., FisherScientific 1 L Sodium Borate Mfr. #159532, or equivalent), or
¾ Phosphate-Buffered Saline (PBS), pH 7.2-7.6 (Sigma® tablets, Mfr. #P4417, or
equivalent) and pH meter or pH indicator strips with pH range 4 to 10
•
Sodium Fluorescein Powder (1-mg vial, BioTek PN 98155)
•
Excitation filter 485/20 nm and Emission filter 528/20 nm installed
•
510 nm dichroic mirror installed
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
101
Fluorescence Polarization (FP) Test
™ The FP Test can be performed in conjunction with the top Corners/
Sensitivity/Linearity Tests, in the same microplate.
•
A new, clean, 96-well solid black microplate, such as Corning® Costar Mfr.
#3915. A Greiner SensoPlate can also be used.
•
The recommended test solutions are available from Invitrogen™ Corporation,
in their “FP One-Step Reference Kit,” (PN P3088) and from BioTek (see page
23). The Invitrogen kit includes:
¾
Green Polarization Reference Buffer, 15 mL
¾
Green Low Polarization Reference, 4 mL
¾
Green High Polarization Reference, 4 mL
¾
The kit also includes two red polarization solutions; these are not used
•
Excitation filter 485/20 nm and Emission filter 528/20 nm installed
•
510 nm dichroic mirror and polarizers installed
Time-Resolved Fluorescence (TRF) Test
•
15 mL conical-bottom, polypropylene sample tube
•
Excitation filter 360/40 nm and Emission filter 620/40 nm installed
•
400 nm dichroic mirror installed
•
A new, clean 96-well solid white microplate, such as Corning Costar Mfr.
#3912.
•
The recommended test solution (FluoSpheres carboxylate-modified
microspheres, 0.2 µm europium luminescent, 2 μL) is available from Invitrogen
Corporation (PN F20881) and from BioTek (see page 23).
Test Solutions
Determine which tests you need to run for your reader model, and then prepare the
necessary solutions. The supplies for each test are listed under “Required Materials”
on page 100.
™ Filter the solutions to remove particulates that could cause erroneous
readings. Do not allow dust to settle on the surface of the solution;
use microplate covers or seals when not reading the plate.
Synergy 2 Operator’s Manual
102 | Chapter 5: Instrument Qualification
Corners/Sensitivity/Linearity (FI) Tests
If using BioTek’s sodium fluorescein powder (PN 98155), be
sure to hold the vial upright and open it carefully; the
material may be concentrated at the top. If a centrifuge is
available, spin down the tube before opening.
When diluting the sodium fluorescein powder in buffer, it
takes time for the powder to completely dissolve. Allow the
solution to dissolve for 4 to 5 minutes, with intermittent
vortexing, before preparing the titration dyes.
Wrap the vial containing the SF stock solution in foil to
prevent exposure to light.
Discard any open, unused solutions after seven days.
1. The Sodium Borate solution described on page 100 does not require further
preparation; proceed to step 2. If you are using PBS, prepare the solution now:
•
(Optional, but recommended) Using a 0.45-micron filter, filter 200 mL of
deionized or distilled water.
•
Follow the manufacturer’s instructions on the PBS packaging to create 200
mL, dissolving the necessary amount of PBS into the filtered water.
•
Stir the solution (preferably using a stir table) until the PBS is completely
dissolved.
•
Check the pH; it should be between 7.2 and 7.6 at 25°C.
2. Prepare the sodium fluorescein stock solution:
•
Add 2.0 mL of the buffer solution to the 1 mg Sodium Fluorescein (SF) vial.
This yields a 1.3288 mM stock solution.
•
Ensure that the dye has completely dissolved and is well mixed.
3. Prepare the dilutions. Label each with “SF” and the concentration:
Mix this SF solution:
with buffer:
to make:
0.53 mL of 1.3288 mM stock solution
13.47 mL
50.2 μM
110 µL of 50.2 μM SF
13.89 mL
400 nM
10.5 mL
100 nM
0.46 mL of 100 nM SF
13.54 mL
3.3 nM
4.24 mL of 3.3 nM SF
9.76 mL
1 nM
3.5 mL of 400 nM SF
Corners Test
Sensitivity/Linearity Tests
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
103
Fluorescence Polarization (FP) Test
The test solutions described on page 101 do not require additional preparation.
Time-Resolved Fluorescence (TRF) Test
•
Shake the FluoSphere® container vigorously for 30 seconds prior to pipetting.
Alternatively, sonicate or vortex the container.
•
Mix 10 µL of FluoSpheres with 10 mL of deionized water, in a 15-mL conicalbottom, polypropylene sample tube. This yields a 20-nM equivalent
suspension.
•
Shake the vial vigorously for 30 seconds prior to pipetting. Alternatively,
sonicate or vortex the container.
•
Mix 10 µL of 20-nM suspension with 10 mL of deionized water, in a 15-mL
conical-bottom, polypropylene sample tube. This yields a 20-pM equivalent
suspension.
•
Refrigerate any unused portions of the FluoSpheres. The temperature must be
between +2°C to +6°C.
™ The prepared TRF plate can be kept for a maximum of 7 days, if
covered and stored in the dark between +2°C to +6°C.
™ Allow the plate to sit at room temperature for approximately 15
minutes prior to use.
™ Shake the plate gently prior to the read.
Synergy 2 Operator’s Manual
104 | Chapter 5: Instrument Qualification
Procedure
1. If you have not already done so, create the Gen5 protocols as described on page 111.
2. If you have not already done so, prepare the solutions for the tests you plan to
perform. See pages 101.
™ Refer to the pipette maps starting on page 105 for steps 3 through 6.
3. Perform the FI tests using the Bottom optics:
•
Pipette the solutions for the Corners, Sensitivity, and Linearity Tests into a
clean 96-well quartz or glass-bottom microplate.
•
Create an experiment based on the FI_B.prt protocol. Read the plate and then
save the experiment.
4. Perform the FI tests using the Top optics:
•
Pipette the solutions for the Corners, Sensitivity, and Linearity Tests into a new
96-well solid black or glass-bottom microplate.
•
Create an experiment based on the FI_T.prt protocol. Read the plate and then
save the experiment.
5. If you are testing FP:
•
Pipette the solutions for the FP test into the same plate as used in step 4.
•
Create an experiment based on the FP.prt protocol. Read the plate and then
save the experiment.
6. If you are testing TRF:
•
Pipette the solutions for the TRF test into a new 96-well solid white plate.
•
Create an experiment based on the TRF.prt protocol. Read the plate and then
save the experiment.
7. Calculate and evaluate results as described under Results Analysis, starting on page
108.
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
105
Pipette Maps
™ Seal the plates with foil or store them in black polyethylene bags until
use. When using a clear-bottom plate, if the base is touched, clean the
entire base with alcohol (95% ethanol) and then wipe with a lint-free
cloth. Before placing a plate in the instrument, blow the bottom of the
plate with an aerosol duster.
The Corners, Sensitivity/Linearity, and FP pipette maps are designed so that multiple
tests can be run using the same plate. For example:
•
Corners, Sensitivity, and Linearity tests for the bottom optics can be performed
using the same quartz or glass-bottom plate.
•
Corners, Sensitivity, Linearity, and FP tests (top optics) can be performed using
the same solid black plate.
Corners Test
™ You can omit the buffer when using a solid black plate or the Greiner
SensoPlate.
•
Pipette 200 µL of the 3.3 nM SF solution into the “corner” wells.
•
Pipette 200 µL of the buffer in the wells surrounding the SF.
1
2
3
4
A
3.3
nM
3.3
nM
3.3
nM
B
BUF
BUF
G
BUF
H
3.3
nM
5
6
7
8
9
10
11
12
BUF
BUF
3.3
nM
3.3
nM
3.3
nM
BUF
BUF
BUF
BUF
BUF
BUF
BUF
BUF
BUF
BUF
BUF
BUF
BUF
3.3
nM
3.3
nM
BUF
BUF
3.3
nM
3.3
nM
3.3
nM
C
D
E
F
Synergy 2 Operator’s Manual
106 | Chapter 5: Instrument Qualification
Sensitivity and Linearity Tests
Use a multi-channel pipette with four tips installed.
Perform these instructions carefully, and refer to the plate map below.
•
Pipette 150 µL of buffer into columns 2-5 and 10-12.
•
Discard the tips.
•
Pipette 150 µL of the 1 nM SF solution into column 1.
•
Pipette 150 µL of the 1 nM SF solution into column 2.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 2 and dispense into column 3.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 3 and dispense into column 4.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 4 and dispense into column 5.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 5. Discard the solution and the tips.
1
2
3
4
5
C
1.0
nM
0.5
nM
0.25
nM
0.125
nM
D
1.0
nM
0.5
nM
0.25
nM
E
1.0
nM
0.5
nM
F
1.0
nM
0.5
nM
6
7
8
9
10
11
12
0.0625
nM
BUF
BUF
BUF
0.125
nM
0.0625
nM
BUF
BUF
BUF
0.25
nM
0.125
nM
0.0625
nM
BUF
BUF
BUF
0.25
nM
0.125
nM
0.0625
nM
BUF
BUF
BUF
A
B
G
H
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
107
FP Test
•
Pipette 200 µL of the green polarization buffer (BUF) into wells A6-H6.
•
Pipette 200 µL of the green high polarization reference (HPR) into wells A7-B7.
•
Pipette 200 µL of the green low polarization reference (LPR) into wells A8-H8.
1
2
3
4
5
6
7
8
A
BUF
HPR
LPR
B
BUF
HPR
LPR
C
BUF
LPR
D
BUF
LPR
E
BUF
LPR
F
BUF
LPR
G
BUF
LPR
H
BUF
LPR
9
10
11
12
TRF Test
•
Pipette 200 µL of deionized water into wells A6-H6.
•
If you have not already done so, shake the vial of 20 pM europium suspension
vigorously for 30 seconds prior to pipetting. Alternatively, sonicate or vortex
the vial.
•
Pipette 200 µL of the 20 pM europium suspension (Eu) into wells A8-B8.
1
2
3
4
5
6
7
8
A
DI
Eu
B
DI
Eu
C
DI
D
DI
E
DI
F
DI
G
DI
H
DI
Synergy 2 Operator’s Manual
9
10
11
12
108 | Chapter 5: Instrument Qualification
Results Analysis
Corners Test
1. Calculate the Mean of the wells containing the 3.3 nM SF test solution (A1-A3,
A10-A12, H1-H3, H10-H12).
2. Calculate the Standard Deviation for the same 12 wells.
3. Calculate the % CV: (Standard Deviation/Mean) * 100
The % CV must be < 3.0 to pass.
Sensitivity Test
1. Calculate the Mean and Standard Deviation for the buffer wells (C10-F12).
2. Calculate the Mean for the 1000 pM (1 nM) SF solution wells (C1-F1).
3. Calculate the Detection Limit, in pM:
1000 / ((Mean SF - Mean Buffer)/(3 * Standard Deviation Buffer))
To pass, the Detection Limit
must be:
Optic Probe
Bottom 5 mm
< 26 pM (10 pg/mL)
Bottom 3 mm
< 53 pM (20 pg/mL)
Bottom 1.5 mm
< 106 pM (40 pg/mL)
Top, with 510 nm dichroic mirror
< 5 pM (2 pg/mL)
Linearity Test
1. Calculate the Mean of the four wells for each concentration in columns 1-5.
2. Perform linear regression using these values as inputs:
x
y
1000
Mean of the 1000 pM wells
500
Mean of the 500 pM wells
250
Mean of the 250 pM wells
125
Mean of the 125 pM wells
62.5
Mean of the 62.5 pM wells
3. Calculate the R-Squared value; it must be >= 0.950 to pass.
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
109
Fluorescence Polarization (FP) Test
1. Using the raw data from the Parallel read:
•
Calculate the Mean Blank (wells A6-H6).
•
Calculate the Signal for each HPR well: Subtract the Mean Blank from
its measurement value.
•
Calculate the Signal for each LPR well: Subtract the Mean Blank from its
measurement value.
2. Using the raw data from the Perpendicular read:
•
Calculate the Mean Blank (wells A6-H6).
•
Calculate the Signal for each HPR well: Subtract the Mean Blank from
its measurement value.
•
Calculate the Signal for each LPR well: Subtract the Mean Blank from its
measurement value.
3. Calculate the G-Factor for each LPR well:
(Parallel LPR Signal * (1-0.02)) / (Perpendicular LPR Signal * (1+0.02))
4. Calculate the Mean G-Factor.
5. Calculate the Polarization value in mP for each HPR well (“PHPR”):
Parallel HPR Signal - Mean G-Factor * Perpendicular HPR Signal
Parallel HPR Signal + Mean G-Factor * Perpendicular HPR Signal
* 1000
6. Calculate the Mean PHPR, in mP. This value must be > 340 mP to pass.
7. Calculate the Polarization value in mP for each LPR well (“PLPR”):
Parallel LPR Signal - Mean G-Factor * Perpendicular LPR Signal
Parallel LPR Signal + Mean G-Factor * Perpendicular LPR Signal
* 1000
8. Calculate the Standard Deviation of the “PLPR,” in mP. This value must be
< 5 mP to pass.
Time-Resolved Fluorescence (TRF) Test
1. Calculate the Mean and Standard Deviation of the wells containing the deionized
water (wells A6-H6).
2. Calculate the Mean and Standard Deviation of the wells containing the
Europium solution (wells A8-B8).
3. Calculate the Detection Limit, in fM:
20000 / ((Mean Eu - Mean DI water)/(3 * Standard Deviation DI water))
The Detection Limit must be < 250 fM to pass.
Synergy 2 Operator’s Manual
110 | Chapter 5: Instrument Qualification
Troubleshooting Tips
If any tests fail, please try the suggestions below. If the test(s) continue to fail, print the
results and contact BioTek’s Technical Assistance Center.
•
Are the solutions fresh? Unless otherwise indicated, once mixed (or opened, if
purchased from the manufacturer), the buffer and stock solutions should be
discarded after seven days.
•
Are the Excitation/Emission filters clean? Are they in the proper locations and
in the proper orientation in the filter wheels?
•
Are you using new/clean plates? We suggest you re-run the test with a
new/clean microplate. If the base of a clear plate is touched, clean the entire
base with alcohol (95% ethanol) and then wipe with a lint-free cloth. Before
placing a plate in the instrument, blow the bottom of the plate with an aerosol.
If the test fails again, the optical probe(s) may need to be cleaned. Contact
BioTek TAC for instructions.
•
When performing the Fluorescence Intensity tests, if a test fails because one or
more wells overranged, reduce the Sensitivity value in the Gen5 protocol by 1-5
counts and re-read the plate.
•
If the Corners Test continues to fail, the hardware may be misaligned. Contact
BioTek TAC.
•
Review the instructions under “Pipette Maps” (starting on page 105) to verify
that you correctly prepared the plates.
•
Does the Plate Type setting in the Gen5 protocol match the plate you used?
•
For injector models, spilled fluid inside the reader may be fluorescing,
corrupting your test results. Clean the internal components according to the
instructions in Chapter 6, Preventive Maintenance, and re-run the tests.
•
When testing the Fluorescence Polarization module using a solid black plastic
microplate, if the standard deviation for the buffer wells is too high, try either
moving the buffer wells to another column, or using the Greiner SensoPlate.
With some black plastic plates, the wells in the center of the plate may be
slightly distorted due to the plate molding process, and this can affect the
standard deviation.
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
111
Gen5 Protocol Reading Parameters
Create Gen5 protocols to test your reader’s configuration:
Protocol
Name
Purpose
FI_B.prt
Fluorescence Intensity tests (Corners, Sensitivity,
Linearity), using the BOTTOM optics
FI_T.prt
Fluorescence Intensity tests (Corners, Sensitivity,
Linearity), using the TOP optics
FP.prt
Fluorescence Polarization test
TRF.prt
Time-Resolved Fluorescence test
The information in the following tables represents the recommended reading
parameters. Your tests may require modification to some of these parameters, such as
the Plate Type or Sensitivity (see Troubleshooting Tips on page 110).
™ The Plate Type setting in each Gen5 protocol should match the plate
you are actually using.
Protocol Name: FI_B.prt
A procedure with two Read steps to test the bottom optics: one for the Corners Test and one
for the Sensitivity/Linearity Tests. All reading parameters are the same for both Read steps,
with the exceptions noted below.
Parameter
Setting
Plate Type:
Read Step Label:
Detection Method:
Read Type:
Read Speed:
Delay After Plate Movement:
Measurements Per Data Point:
Read Wells:
Light Source:
Filter Sets:
Filters:
Optics Position:
Sensitivity:
Synergy 2 Operator’s Manual
“Greiner SensoPlate” (#655892)
Corners Read step: “Corners Read”
Sensitivity/Linearity Read step: “Sensitivity Read”
Fluorescence
Endpoint
Normal
350 msec
40
Corners Read step: Full plate
Sensitivity/Linearity Read step: Wells C1 to F12
Tungsten
1
EX 485/20 nm, EM 528/20 nm
Bottom
Corners Read step: 80
Sensitivity/Linearity Read step: 100
112 | Chapter 5: Instrument Qualification
Protocol Name: FI_T.prt
A procedure with two Read steps to test the top optics: one for the Corners Test and one for
the Sensitivity/Linearity Tests. All reading parameters are the same for both Read steps, with
the exceptions noted below.
Parameter
Setting
Plate Type:
Read Step Label:
Detection Method:
Read Type:
Read Speed:
Delay After Plate Movement:
Measurements Per Data Point:
Read Wells:
Light Source:
Filter Sets:
Filters:
Optics Position:
Sensitivity:
Top Probe Vertical Offset:
“Costar 96 black opaque” (#3915)
Corners Read step: “Corners Read”
Sensitivity/Linearity Read step: “Sensitivity Read”
Fluorescence
Endpoint
Normal
350 msec
40
Corners Read step: Full plate
Sensitivity/Linearity Read step: Wells C1 to F12
Tungsten
1
EX 485/20 nm, EM 528/20 nm
Top 510 nm
Corners Read step: 75
Sensitivity/Linearity Read step: 75
5.00 mm
Protocol Name: FP.prt
A procedure with one Read step with Polarization enabled, inside a Plate Mode block.
Parameter
Setting
Plate Type:
Synchronized Mode:
Detection Method:
Read Type:
Read Speed:
Delay After Plate Movement:
Measurements Per Data Point:
“Costar 96 black opaque” (#3915)
Plate Mode with Timing Control
Fluorescence
Endpoint
Normal
350 msec
60
Read Wells:
A5-H9
Polarization:
Enabled
Light Source:
Filters:
Optics Position:
Sensitivity:
Top Probe Vertical Offset:
Tungsten
EX 485/20 nm, EM 528/20 nm
Top 510 nm
100
5.00 mm
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
Protocol Name: TRF.prt
A procedure with one Read step with Time-Resolved enabled.
Parameter
Setting
Plate Type:
“Costar 96 white opaque” (#3912)
Delay Step:
3 minutes
Shake Step:
30 seconds at Medium intensity
Detection Method:
Read Type:
Read Speed:
Delay After Plate Movement:
Measurements Per Data Point:
Read Wells:
Time-Resolved:
Delay Before Collecting Data:
Data Collection Time:
Light Source:
Filter Sets:
Filters:
Optics Position:
Sensitivity:
Top Probe Vertical Offset:
Synergy 2 Operator’s Manual
Fluorescence
Endpoint
Normal
350 msec
20
A5-H9
Enabled
300 µsec
1000 µsec
Xenon Flash
1
EX 360/40 nm, EM 620/40 nm
Top 400 nm
125
5.00 mm
113
114 | Chapter 5: Instrument Qualification
Fluorescence Tests Using Methylumbelliferone
As an alternative to using Sodium Fluorescein, Methylumbelliferone (“MUB”) can be
used to test the top optics.
Required Materials
™ BioTek offers test kits containing the microplates and solutions used in
fluorescence liquid test procedures; see page 23.
™ Microplates should be perfectly clean and free from dust or bottom
scratches. Use new microplates from sealed packages.
™ Manufacturer part numbers are subject to change.
•
Methylumbelliferone (“MUB”) (10-mg vial, BioTek PN 98156)
•
Carbonate-Bicarbonate buffer (“CBB”) capsules (BioTek PN 98158)
•
100% methanol (BioTek PN 98161)
•
A new, clean, 96-well solid black microplate, such as Corning® Costar #3915 (or
equivalent)
•
Excitation filter 360/40 nm, Emission filter 460/40 nm
•
50% mirror installed
•
Deionized or distilled water
•
Various beakers, graduated cylinders, and pipettes
•
Aluminum foil
•
(Optional, but recommended) 0.45 micron filter
•
(Optional) Black polyethylene bag(s) to temporarily store plate(s)
•
Gen5 protocol FI_MUB.prt described on page 118
Test Solutions
Filter solutions to remove particulates that could cause
erroneous readings. Do not allow dust to settle on the
surface of the solution; use microplate covers or seals
when not reading the plate.
Wrap the vial containing the MUB stock solution in foil
to prevent exposure to light.
Discard any open, unused solutions after seven days.
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
115
1. Prepare the buffer (CBB) solution:
•
(Optional, but recommended) Using a 0.45-micron filter, filter 200 mL of
deionized or distilled water.
•
Open and dissolve the contents of 2 CBB capsules (do not dissolve the
outer gelatin capsule) into 200 mL of the water.
•
Stir the solution (preferably using a stir table) until the CBB is
completely dissolved.
2. Prepare the MUB stock solution:
•
Add 1 mL of 100% methanol to the 10 mg vial of MUB.
•
Make sure all of the dye has completely dissolved and is well mixed.
This yields a 10 mg/mL stock solution.
•
Wrap the solution in aluminum foil to prevent exposure to light.
3. Prepare the dilutions. Label each with “MUB” and the concentration:
Mix this MUB solution:
with:
to make:
0.5 mL of 10 mg/mL stock solution
4.5 mL of 100%
methanol
1 mg/mL
0.88 mL of 1 mg/mL solution
4.12 mL of CBB
176 μg/mL
0.1 mL of 176 μg/mL solution
9.9 mL of CBB
1.76 μg/mL
0.5 mL of 1.76 μg/mL solution
4.5 mL of CBB
176 ng/mL
9 mL of CBB
17.6 ng/mL
(100 nM)
1 mL of 176 ng/mL solution
Procedure
1. If you have not already done so, prepare the test solutions. See page 114.
2. Perform the Sensitivity/Linearity tests using the Top optics:
•
Refer to Pipette Map on the next page and pipette the solutions into a
clean, 96-well solid black plate.
•
Create a Gen5 experiment based on the FI_MUB.prt protocol and read
the plate.
3. Calculate and evaluate results as described under Results Analysis on page 117.
Synergy 2 Operator’s Manual
116 | Chapter 5: Instrument Qualification
Pipette Map
™ Seal the plate with foil or store it in black polyethylene bags until use.
Use a multi-channel pipette with 4 tips installed to process rows C-F:
•
Pipette 150 µL of buffer into columns 10-12.
•
Pipette 150 µL of buffer into columns 2-5 (not column 1).
•
Discard the tips.
•
Pipette 150 µL of the 17.6 ng/mL (100 nM) MUB solution into column 1.
Discard the tips.
•
Pipette 150 µL of the 17.6 ng/mL (100 nM) MUB solution into column 2.
Do not discard the tips.
•
Aspirate 150 µL from column 2 and dispense it into column 3.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 3 and dispense it into column 4.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 4 and dispense it into column 5.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 5 and discard the fluid and tips.
1
2
3
4
5
C
100
nM
50
nM
25
nM
12.5
nM
D
100
nM
50
nM
25
nM
E
100
nM
50
nM
F
100
nM
50
nM
6
7
8
9
10
11
12
6.25
nM
BUF
BUF
BUF
12.5
nM
6.25
nM
BUF
BUF
BUF
25
nM
12.5
nM
6.25
nM
BUF
BUF
BUF
25
nM
12.5
nM
6.25
nM
BUF
BUF
BUF
A
B
G
H
BioTek Instruments, Inc.
Fluorescence Liquid Tests |
117
Results Analysis
Sensitivity Test
1. Calculate the Mean and Standard Deviation for the buffer wells (C10-F12).
2. Calculate the Mean for the 17.6 ng/mL (100 nM) MUB solution wells (C1-F1).
3. Calculate the Detection Limit, in ng/mL:
17.6 / ((Mean MUB - Mean Buffer)/(3 * Standard Deviation Buffer))
For the Top probe with the 50% mirror, the Detection Limit must be
< 0.16 ng/mL to pass.
Linearity Test
1. Calculate the Mean of the four wells for each concentration in columns 1-5.
2. Perform linear regression using these values as inputs:
x
y
100
Mean of the 100 nM wells
50
Mean of the 50 nM wells
25
Mean of the 25 nM wells
12.5
Mean of the 12.5 nM wells
6.25
Mean of the 6.25 nM wells
3. Calculate the R-Squared value; it must be >= 0.950 to pass.
Synergy 2 Operator’s Manual
118 | Chapter 5: Instrument Qualification
Gen5 Protocol Reading Parameters
The information below represents the recommended reading parameters. Your tests
may require modification to some of these parameters, such as the Plate Type or
Sensitivity (see Troubleshooting Tips on page 110).
™ The Plate Type setting in the Gen5 protocol should match the plate
you are actually using.
Protocol Name: FI_MUB.prt
Parameter
Setting
Plate Type:
Detection Method:
Read Type:
Read Speed:
Delay After Plate Movement:
Measurements Per Data Point:
Dynamic Range:
Read Wells:
Light Source:
Filter Sets:
Filters:
Optics Position:
Sensitivity:
Top Probe Vertical Offset:
“Costar 96 black opaque” (#3915)
Fluorescence
Endpoint
Normal
350 msec
40
Standard
C1-F12
Tungsten
1
EX 360/40 nm, EM 460/40 nm
Top 50% Mirror
90
5.00 mm
BioTek Instruments, Inc.
119
Luminescence Test |
Luminescence Test
This section only applies to models with the Luminescence module.
For Synergy 2 models with luminescence capability, BioTek provides two methods for
verifying the performance of the luminescence module.
One method measures a Harta Luminometer Reference Microplate, which is an LED-based
test plate. Contact BioTek to purchase a plate, or go to www.hartainstruments.com for
more information. The other method measures a LUX Biotechnology, Ltd., Glowell unit,
which is a small, sealed cylinder with a gaseous tritium light source.
Harta Plate Test
Materials
•
Luminometer Reference Microplate and Adapter, BioTek PN 8030015
•
Gen5 protocol LumTest_Harta.prt (see page 123)
Procedure
1. Turn on the Harta reference plate using the I/O switch on the back of the plate.
2. Check the plate’s battery by pressing simultaneously on the two test buttons on
the back of the plate and ensuring that the test light turns on.
3. Place the Harta plate adapter on the reader’s carrier, then place the test plate on
top of the adapter.
4. Create an experiment based on the LumTest_Harta.prt protocol and read the
plate.
5. Calculate and evaluate results as described under Results Analysis on page 120.
Plate Map
1
2
3
4
5
6
A2
meas
A
7
8
battery
check
battery
check
9
10
11
12
B
C
D
E
F
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
G
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
buffer
H
Synergy 2 Operator’s Manual
120 | Chapter 5: Instrument Qualification
Results Analysis
™ A manual ATP correlation process determined that 11,000 RLU from
the Harta plate is equivalent to approximately 1800 attomoles of ATP.
1. On the Harta plate’s Calibration Certificate, locate the NIST measurement for the
A2 position and convert it to attomoles:
(A2 NIST measurement/11,000)*1800
2. Determine if the plate’s battery is still functioning properly:
•
If A8 > A7, the battery is good
•
If A8 < A7, the battery requires replacement
3. Calculate the signal-to-noise ratio:
(A2 – Mean of the buffer cells)/(3 * Standard deviation of buffer cells)
4. Calculate the detection limit:
A2 NIST measurement in attomoles/signal-to-noise ratio
•
If the reader is equipped with the low-noise PMT, the detection limit must be
< 50 amol for the test to pass.
•
If the reader is equipped with the red-shifted PMT, the detection limit must be
< 500 amol for the test to pass.
•
To determine which PMT is installed, check the label on the side of the reader.
#49984 = low noise PMT, #49721 = red-shifted.
Glowell Test
Materials
•
Glowell, PN GLO-466, formerly available from LUX BioTechnology, Ltd.
•
Glowell Adapter Plate, available from BioTek, PN 7160006
•
Gen5 protocol LumTest.prt (see page 122)
Procedure
1. If you have not already done so, insert the Glowell (“window” side up) into well
D8 of the Adapter Plate.
2. If you have not already done so, create the Gen5 protocol as described on page
122.
3. Create an experiment based on the LumTest.prt protocol. Read the plate and
then save the experiment.
4. Calculate and evaluate results as described under Results Analysis on page 121.
BioTek Instruments, Inc.
Luminescence Test |
121
Results Analysis
™ A manual ATP correlation process determined that 0.021 pW Radiant
Flux is equivalent to approximately 1800 attomoles of ATP.
1. Locate these items on the Glowell’s Calibration Certificate: Calibration Date,
Radiant Flux (pW), Measurement Uncertainty of the Radiant Flux.
2. Calculate the number of days between the Calibration Date and the date the test
was performed.
3. Correct the Glowell’s Radiant Flux value for deterioration over time:
Radiant Flux * e^(-0.0001536*number of days since calibration)
4. Convert the Corrected Radiant Flux value to attomoles:
(Corrected Radiant Flux / 0.021) * 1800
5. Calculate an error factor for the Corrected Radiant Flux (amol):
(Corrected Radiant Flux in amol * Measurement Uncertainty) / 100
6. Calculate the min/max criteria for the Corrected Radiant Flux (amol):
MIN: Corrected Radiant Flux in amol – Error Factor
MAX: Corrected Radiant Flux in amol + Error Factor
7. Calculate the Signal-to-Noise Ratio:
Measurement value of the Glowell – Mean of Column 9
3 x Standard Deviation of Column 9
8. Calculate the Detection Limit:
Corrected Radiant Flux in amol / Signal-to-Noise Ratio
9. Calculate the min/max criteria for the Detection Limit:
MIN: MIN for Corrected Radiant Flux in amol / Signal-to-Noise Ratio
MAX: MAX for Corrected Radiant Flux in amol / Signal-to-Noise Ratio
•
If the reader is equipped with the low-noise PMT, both of these values must
be < 50 amol for the test to pass.
•
If the reader is equipped with the red-shifted PMT, both of these values
must be < 500 amol for the test to pass.
•
To determine which PMT is installed, check the label on the side of the
reader. #49984 = low noise PMT, #49721 = red-shifted.
Synergy 2 Operator’s Manual
122 | Chapter 5: Instrument Qualification
Troubleshooting
If the test fails, please try the suggestions below. If the tests continue to fail, print the
results and contact BioTek’s Technical Assistance Center.
•
Ensure that the reading is performed through a hole in the EM filter wheel, not
through a glass filter.
•
Verify that the filter wheel settings in Gen5 match the physical wheel.
•
If the test continues to fail, the optical probe(s) may need to be cleaned. Contact
BioTek TAC for instructions.
Glowell only:
•
Is the Glowell properly inserted into the adapter? The “window” side should
be facing up. If necessary, clean the Glowell according to the manufacturer’s
instructions.
•
Is the adapter plate clean? If dust has collected in the wells, try cleaning the
plate using compressed air or an aerosol duster.
•
Is the test failing because the standard deviation of the empty background
wells is 0 (resulting in a division-by-zero error when calculating the Signal-toNoise Ratio)? If yes, try pipetting 100 µL of deionized water into all wells of
Columns 9, 10, and 11 (the background wells).
Gen5 Protocol Reading Parameters
The information in the following tables represents the recommended reading
parameters.
Protocol Name: LumTest.prt (Glowell)
Parameter
Setting
Plate Type:
“Costar 96 black opaque”
Delay Step:
3 minutes
Detection Method:
Read Type:
Integration Time:
Luminescence
Endpoint
0:10.00 MM:SS:ss
Delay After Plate Movement:
350 msec
Dynamic Range:
Standard
Read Wells:
Emission:
A8-A11
Hole
Optics Position:
Top
Sensitivity:
150
Top Probe Vertical Offset:
1.00 mm
BioTek Instruments, Inc.
Luminescence Test |
Protocol Name: LumTest_Harta.prt
Parameter
Setting
Plate Type:
“Costar 96 black opaque”
Delay Step:
3 minutes
READ STEP 1
Label:
Read Wells:
Detection Method:
Read Type:
Integration Time:
“Reference well A2”
A2
Luminescence
Endpoint
0:10.00 MM:SS:ss
Delay After Plate Movement:
350 msec
Dynamic Range:
Standard
Emission:
Hole
Optics Position:
Top
Sensitivity:
150
Top Probe Vertical Offset:
5.00 mm
READ STEP 2
Label:
Read Wells:
Detection Method:
Read Type:
Integration Time:
“Background”
F1-G12
Luminescence
Endpoint
0:10.00 MM:SS:ss
Delay After Plate Movement:
350 msec
Dynamic Range:
Standard
Emission:
Hole
Optics Position:
Top
Sensitivity:
150
Top Probe Vertical Offset:
4.00 mm
READ STEP 3
Label:
Read Wells:
Detection Method:
Read Type:
Integration Time:
“Battery Check”
A7-A8
Luminescence
Endpoint
0:01.00 MM:SS:ss
Delay After Plate Movement:
350 msec
Dynamic Range:
Standard
Emission:
Optics Position:
Sensitivity:
Top Probe Vertical Offset:
Synergy 2 Operator’s Manual
Hole
Top
50
5.00 mm
123
124 | Chapter 5: Instrument Qualification
Dispense Module Tests
This section only applies to models with injectors.
BioTek developed a set of tests that you can perform to ensure that the dispense module
performs to specification initially and over time. We recommend that you perform these
tests before first use (e.g., during the Initial OQ), and then every three months.
•
The Accuracy Test is a measure of the mean volume per well for multiple
dispenses. The actual weight of the dispensed fluid is compared to the expected
weight and must be within a certain percentage to pass. Pass/Fail criteria depends
on the per-well volume dispensed: 2.0% for 80 µL, 5.0% for 20 µL, and 20.0% for
5 µL. It is assumed that one gram is equal to one milliliter.
The test uses a green dye test solution and one 96-well microplate (per injector) to
test the three different volumes. The balance is tared with the empty plate, and
then the 80 µL dispense is performed for columns 1-4. The fluid is weighed and the
balance is tared again (with the plate on the balance). This process is repeated for
the 20 µL and 5 µL dispenses.
•
The Precision Test is a measure of the variation among volumes dispensed to
multiple wells. For each volume dispensed (80/20/5 µL) to four columns, the
coefficient of variation of 32 absorbance readings is calculated. Pass/Fail criteria
depends on the per-well volume dispensed: 2.0% for 80 µL, 7.0% for 20 µL, and
10.0% for 5 µL. The plate is read in an absorbance reader at 405/750 nm for
columns 1-4 and at 630/750 nm for columns 5-12.
The two tests are performed simultaneously and use the same plate.
Each dispense module is calibrated to perform with a specific reader.
Make sure the dispense module and reader have the same serial number.
Required Materials
•
Absorbance reader with 405, 630, and 750 nm filters. The reader must have an
accuracy specification of ± 1.0% ± 0.010 OD or better and a repeatability
specification of ± 1.0% ± 0.005 OD or better.
™ The Synergy 2 may be used if it is equipped with the Absorbance
module and has passed the Absorbance Plate Test and the
Absorbance Liquid Tests.
•
Microplate shaker (if the absorbance reader does not support shaking)
•
Precision balance with capacity of 100 g minimum and readability of 0.001 g
BioTek Instruments, Inc.
Dispense Module Tests |
125
•
50-200 µL hand pipette and disposable tips
•
Deionized water
•
Supply bottles
•
250 mL beaker
•
New 96-well, clear, flat-bottom microplates
•
BioTek’s Green Test Dye Solution (PN 7773003) undiluted, or one of the alternate
test solutions listed in the next section
•
100 mL graduated cylinder and 10 mL pipettes (if not using BioTek’s test solution)
•
Gen5 protocols as defined by the procedure on page 129 or 131
Test Solutions
™ 80 µL of test solution with 150 µL of deionized water should read
between 1.300 and 1.700 OD at 405/750 nm. The solutions should be
at room temperature.
If you do not have BioTek’s Green Test Dye Solution (PN 7773003), prepare a green test
dye solution using one of the following methods:
•
Using BioTek’s Blue and Yellow Concentrate Dye Solutions:
Ingredient
Concentrate Blue Dye Solution (PN 7773001; 125 mL)
4.0 mL
QC (Yellow) Solution (PN 7120782; 125 mL)
5.0 mL
Deionized water
•
Quantity
90.0 mL
Using FD&C Blue and Yellow Dye Powder:
Ingredient
Quantity per Liter
FD&C Blue #1
0.200 g
FD&C Yellow #5
0.092 g
Tween
®
20
1.0 mL
Sodium Azide N3Na
0.010 g
Deionized water
Synergy 2 Operator’s Manual
Make to 1 liter
126 | Chapter 5: Instrument Qualification
Procedure for Models with the Absorbance Module
1. If you have not already done so, create Gen5 protocols Disp 1 Test.prt and Disp 2
Test.prt. Instructions begin on page 129.
2. Prime both dispensers with 4000 µL of deionized or distilled water.
3. Remove the inlet tubes from the supply bottles. Prime both dispensers with the
Volume set to 2000 µL. This prevents the water from diluting the dye.
4. Fill a beaker with at least 20 mL of the green dye solution. Prime both dispensers
with 2000 µL of the solution. When finished, remove the priming plate from the
carrier.
5. In Gen5, create an experiment based on the Disp 1 Test protocol.
6. Place a new 96-well microplate on the balance and tare the balance.
7. Place the plate on the microplate carrier.
™ When each dispense step is finished, you will weigh the plate, record
the weight, tare the balance with the plate on it, and then place the
plate back on the carrier for the next step.
8. Select Plate > Read and click READ. Gen5 will prompt you to empty the tip
priming trough.
9. When ready, click OK to begin the experiment. The sequence is as follows:
•
Dispense 80 µL/well to columns 1-4.
•
Remove the plate and weigh it. Record the weight and tare the balance.
•
Place the plate on the carrier, dispense 20 µL/well to columns 5-8.
•
Remove the plate and weigh it. Record the weight and tare the balance.
•
Place the plate on the carrier, dispense 5 µL/well to columns 9-12.
•
Remove the plate and weigh it. Record the weight.
•
Manually pipette 150 µL of deionized or distilled water into all 12 columns,
on top of the green test dye solution.
•
Place the plate on the carrier for a 15-second shake, the ’80 µL’ read at
405/750 nm, and the ’20 and 5 µL’ read at 630/750 nm.
10. When processing is complete, select File > Save As. Enter an identifying file name
and click Save.
11. Remove the plate from the carrier and set it aside.
12. Repeat steps 5-11 using the Disp 2 Test protocol.
13. See page 128 for instructions for analyzing the results.
14. When all tests are complete, prime both dispensers with at least 5000 µL of deionized
water, to flush out the green dye solution.
BioTek Instruments, Inc.
Dispense Module Tests |
127
Procedure for Models without the Absorbance Module
™ If you will not be using a BioTek absorbance reader for this procedure,
prepare your reader to perform two reads with these characteristics:
‘80 µL’ Read
‘20 & 5 µL’ Read
Primary wavelength:
405 nm
630 nm
Reference wavelength:
750 nm
750 nm
1-4
5-12
Plate columns:
1. If you have not already done so, create the necessary Gen5 protocols as described on
page 131.
2. Prime both dispensers with 4000 µL of deionized or distilled water.
3. Remove the inlet tubes from the supply bottles. Prime both dispensers with the
Volume set to 2000 µL. This prevents the water from diluting the dye.
4. Fill a beaker with at least 20 mL of the green dye solution. Prime both dispensers
with 2000 µL of the solution. When finished, remove the priming plate from the
carrier.
5. In Gen5, create an experiment based on the Disp 1 Test protocol.
6. Place a new 96-well microplate on the balance and tare the balance.
7. Place the plate on the microplate carrier.
™ When each dispense step is finished, you will weigh the plate, record
the weight, tare the balance with the plate on it, and then place the
plate back on the carrier for the next step.
8. Select Plate > Read and click READ. Gen5 will prompt you to empty the tip
priming trough.
9. When ready, click OK to begin the experiment. The sequence is as follows:
•
Dispense 80 µL/well to columns 1-4.
•
Remove the plate and weigh it. Record the weight and tare the balance.
•
Place the plate on the carrier, dispense 20 µL/well to columns 5-8.
•
Remove the plate and weigh it. Record the weight and tare the balance.
•
Place the plate on the carrier, dispense 5 µL/well to columns 9-12.
•
Remove the plate and weigh it. Record the weight.
•
Manually pipette 150 µL of deionized or distilled water into all 12 columns,
on top of the green test dye solution.
•
Carefully set the plate aside.
Synergy 2 Operator’s Manual
128 | Chapter 5: Instrument Qualification
10. Close the experiment without saving it.
™ If you are not using a BioTek reader for the absorbance measurements,
read the plate using the wavelengths shown in the table on the previous
page and then proceed to Results Analysis below.
11. Configure Gen5 to communicate with the BioTek absorbance reader.
12. Create an experment based on the Disp 1 Test Other Reader protocol.
13. Select Plate > Read and click READ. Place the plate on the carrier and click OK.
The absorbance reader will:
•
Shake the plate for 15 seconds.
•
Perform the ’80 µL’ read at 405/750 nm.
•
Perform the ’20 and 5 µL’ read at 630/750 nm.
14. When processing is complete, select File > Save As. Enter an identifying file name
and click Save.
15. Repeat steps 5-14 using the Disp 2 Test protocol for the dispense portion.
16. See the instructions below for analyzing the results.
17. When all tests are complete, prime both dispensers with at least 5000 µL of deionized
water, to flush out the green dye solution.
Results Analysis
™ Worksheets are included at the end of this chapter for recording the
dispense weights, Delta OD values, calculations, and pass/fail.
For each volume dispensed (80, 20, 5 µL), for each dispenser (1, 2):
•
Calculate the Standard Deviation of the 32 wells
•
Calculate the Mean of the 32 wells
•
Calculate the %CV: (Standard Deviation / Mean) x 100
•
Calculate the Accuracy % Error:
((Actual Weight - Expected Weight) / Expected Weight) * 100
™ Expected Weights for 32 wells: 80 μL (2.560 g), 20 μL (0.640 g), 5 μL
(0.160 g). It is assumed that one gram is equal to one milliliter.
Dispense Volume
To pass, %CV
must be:
To pass, Accuracy %
Error must be:
80 µL
<= 2.0%
<= 2.0%
20 µL
<= 7.0%
<= 5.0%
5 µL
<= 10.0%
<= 20.0%
BioTek Instruments, Inc.
Dispense Module Tests |
129
Failures
If any tests fail, prime the fluid lines and re-run the test(s). If the test(s) fail again:
•
The injector heads may require cleaning (see Chapter 6, Preventive
Maintenance).
•
Each dispense module is factory-calibrated for the reader it ships with.
Verify that the serial number on the dispense module matches the serial
number on the reader. Even if the serial numbers match, it is still possible
that the calibration values have been inadverently changed. Contact
BioTek’s Technical Assistance Center.
If tests continue to fail, contact BioTek’s Technical Assistance Center.
Gen5 Test Protocols for Models with the Absorbance Module
™ Perform these steps to create a protocol to test Dispenser 1. Then,
open a copy of the protocol and change the relevant Procedure
parameters for Dispenser 2.
1. In Gen5, create a new Synergy 2 protocol.
2. Define the Procedure with the steps and settings as described in this table:
#
Step Type
1
Dispense
2
Plate Out,In
3
Dispense
4
Plate Out,In
5
Dispense
6
Plate Out,In
7
Shake
Synergy 2 Operator’s Manual
Details
Dispenser <select 1 or 2, depending on the protocol>
Dispense to wells A1..H4
Tip prime before this dispense step, 20 µl
Dispense 80 µl at rate 275 µl/sec
Suggested comment: Weigh the plate (80 ul test). RECORD the
weight, TARE the balance. Place the plate back on the carrier. Click
OK to continue.
Dispenser <select 1 or 2, depending on the protocol>
Dispense to wells A5..H8
Tip prime before this dispense step, 20 µl
Dispense 20 µl at rate 250 µl/sec
Suggested comment: Weigh the plate (20 ul test). RECORD the
weight, TARE the balance. Place the plate back on the carrier. Click
OK to continue.
Dispenser <select 1 or 2, depending on the protocol>
Dispense to wells A9..H12
Tip prime before this dispense step, 5 µl
Dispense 5 µl at rate 225 µl/sec
Suggested comment: Weigh the plate (5 ul test). RECORD the
weight. PIPETTE 150 ul/well of DI water into all 12 columns. Place
the plate back on the carrier. Click OK to perform the Read steps.
Medium intensity for 15 seconds
130 | Chapter 5: Instrument Qualification
#
Step Type
Details
8
Read
Step label:
Wells:
Detection Method:
Read Type:
Read Speed:
Two Wavelengths:
“80 ul Read_Disp 1” (or _Disp 2)
A1..H4
Absorbance
Endpoint
Normal
405 and 750 nm
9
Read
Step label:
Wells:
Detection Method:
Read Type:
Read Speed:
Two Wavelengths:
“20 and 5 ul Read_Disp 1” (or _Disp 2)
A5..H12
Absorbance
Endpoint
Normal
630 and 750 nm
3. Create Data Reduction steps to calculate Delta OD values:
•
Open the Data Reduction dialog and click Transformation.
•
Click Select Multiple Data Sets and then click DS2.
•
Set the Data In for DS1 to the 80 µL Read step at 405 nm.
•
Set the Data In for DS2 to the 80 µL Read step at 750 nm.
•
Click OK to return to the Transformation dialog.
•
In the New Data Set Name field, type an identifying name such as
‘Delta OD 80 ul_Disp 1’.
•
Clear Use single formula for all wells.
•
In the Current Formula field, type DS1-DS2 and then highlight wells
A1 to H4 to assign the formula.
•
Click OK to add the transformation to the Data Reduction list.
•
Create another Transformation similar to the above, with these
characteristics:
¾ DS1 set to the 20 and 5 µL Read step at 630 nm
¾ DS2 set to the 20 and 5 µL Read step at 750 nm
¾ New Data Set Name resembling ‘Delta OD 20 and 5 ul_Disp 1’
¾ Remember to clear ‘Use Single Formula…’
¾ Formula DS1-DS2 applied to wells A5 to H12
4. (This step is optional.) The results analysis worksheet at the end of this chapter
requires the calculation of the Standard Deviation, Mean, and % CV of the ODs
read for each dispense volume in each plate (six sets of calculations). By
identifying the wells by their dispense volumes in the Plate Layout, Gen5 will
calculate these values for you.
•
Open the Plate Layout dialog.
BioTek Instruments, Inc.
Dispense Module Tests |
131
•
Define three Assay Control names as Disp_80, Disp_20, and Disp_5.
•
Assign Disp_80 to wells A1 to H4.
•
Assign Disp_20 to wells A5 to H8.
•
Assign Disp_5 to wells A9 to H12.
™ After running the experiment, view the Statistics for each Delta OD
Data Set to view the calculations.
5. Save the protocols as Disp 1 Test.prt and Disp 2 Test.prt.
Gen5 Test Protocols for Models without the Absorbance Module
The test procedure on page 127 dispenses three volumes of fluid to a microplate and
then reads the plate on an absorbance reader. The procedure is performed twice, once
for each dispenser. You will create two Gen5 protocols to perform the dispense steps. If
you will use a BioTek absorbance reader that is supported by Gen5, you will create one
additional protocol to perform the Read steps.
Create the Dispense Protocols
™ Perform these steps to create a protocol to test Dispenser 1. Then,
open a copy of the protocol and change the relevant Procedure
parameters for Dispenser 2.
1. In Gen5, create a new Synergy 2 protocol.
2. Define the Procedure with the steps and settings as described in this table:
#
Step Type
1
Dispense
Dispenser <select 1 or 2, depending on the protocol>
Dispense to wells A1..H4
Tip prime before this dispense step, 20 µl
Dispense 80 µl at rate 275 µl/sec
2
Plate Out,In
Suggested comment: Weigh the plate (80 ul test).
RECORD the weight, TARE the balance. Place the plate
back on the carrier. Click OK to continue.
3
Dispense
Dispenser <select 1 or 2, depending on the protocol>
Dispense to wells A5..H8
Tip prime before this dispense step, 20 µl
Dispense 20 µl at rate 250 µl/sec
4
Plate Out,In
Suggested comment: Weigh the plate (20 ul test).
RECORD the weight, TARE the balance. Place the plate
back on the carrier. Click OK to continue.
5
Dispense
Dispenser <select 1 or 2, depending on the protocol>
Dispense to wells A9..H12
Tip prime before this dispense step, 5 µl
Dispense 5 µl at rate 225 µl/sec
Synergy 2 Operator’s Manual
Details
132 | Chapter 5: Instrument Qualification
#
Step Type
6
Plate Out,In
7
Read
Details
Suggested comment: Weigh the plate (5 ul test).
RECORD the weight. Set the plate aside and click OK.
Wells:
Detection Method:
Read Type:
Read Speed:
Wavelength:
A1
<select any valid method>
Endpoint
Normal
<select any valid wavelength(s)>
The Read step is necessary because Gen5 requires a Read step within any
Dispense procedure. When the test is run, the measurement value is not used.
3. Save the protocols as Disp 1 Test.prt and Disp 2 Test.prt.
Create the Read Protocol (if needed)
1. In Gen5, create a new Synergy 2 protocol.
2. Define the Procedure with the steps and settings as described in this table:
#
Step Type
Details
1
Shake
2
Read
Step label:
Wells:
Detection Method:
Read Type:
Read Speed:
Two Wavelengths:
“80 ul Read”
A1..H4
Absorbance
Endpoint
Normal
405 and 750 nm
3
Read
Step label:
Wells:
Detection Method:
Read Type:
Read Speed:
Two Wavelengths:
“20 and 5 ul Read”
A5..H12
Absorbance
Endpoint
Normal
630 and 750 nm
Medium intensity for 15 seconds
3. Create Data Reduction steps to calculate Delta OD values:
•
Open the Data Reduction dialog and click Transformation.
•
Click Select Multiple Data Sets and then click DS2.
•
Set the Data In for DS1 to the 80 µL Read step at 405 nm.
•
Set the Data In for DS2 to the 80 µL Read step at 750 nm.
•
Click OK to return to the Transformation dialog.
•
In the New Data Set Name field, type an identifying name such as
‘Delta OD 80 ul_Disp 1’.
•
Clear Use single formula for all wells.
BioTek Instruments, Inc.
Dispense Module Tests |
133
•
In the Current Formula field, type DS1-DS2 and then highlight wells
A1 to H4 to assign the formula.
•
Click OK to add the transformation to the Data Reduction list.
•
Create another Transformation similar to the above, with these
characteristics:
¾ DS1 set to the 20 and 5 µL Read step at 630 nm
¾ DS2 set to the 20 and 5 µL Read step at 750 nm
¾ New Data Set Name resembling ‘Delta OD 20 and 5 ul_Disp 1’
¾ Remember to clear ‘Use Single Formula…’
¾ Formula DS1-DS2 applied to wells A5 to H12
4. (This step is optional.) The results analysis worksheet at the end of this chapter
requires the calculation of the Standard Deviation, Mean, and % CV of the ODs
read for each dispense volume in each plate (six sets of calculations). By
identifying the wells by their dispense volumes in the Plate Layout, Gen5 will
calculate these values for you.
•
Open the Plate Layout dialog.
•
Define three Assay Control names as Disp_80, Disp_20, and Disp_5.
•
Assign Disp_80 to wells A1 to H4.
•
Assign Disp_20 to wells A5 to H8.
•
Assign Disp_5 to wells A9 to H12.
™ After running the experiment, view the Statistics for each Delta OD
Data Set to view the calculations.
5. Save the protocol as Disp Test Other Reader.prt.
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BioTek Instruments, Inc.
†P
Comments:
Signature:
Must be <= 7.0%
Reading Date:
†F
Tested By:
†P
Reader S/N:
Reader Model:
Must be <= 2.0%
%CV:
%CV:
%
Mean:
Mean:
Must be <= 5.0%
Accuracy % Error:
Standard Deviation:
†F
%
Expected weight:
Standard Deviation:
Must be <= 2.0%
Accuracy % Error:
2.5600 g
Expected weight:
20 µL weight:
†P
†P
†F
†F
%
%
0.6400 g
g
Signature:
Reviewed/
Approved By:
Must be <= 10.0%
%CV:
Mean:
Standard Deviation:
Must be <= 20.0%
Accuracy % Error:
Expected weight:
5 µL weight:
†P
†P
†F
†F
%
%
0.1600 g
g
H
H
g
G
G
80 µL weight:
F
12
F
11
E
10
E
9
D
8
D
7
C
6
C
5
B
4
B
3
5 µL Dispense
Delta ODs @630/750 nm
A
2
20 µL Dispense
Delta ODs @630/750 nm
A
1
80 µL Dispense
Delta ODs @405/750 nm
Synergy 2 Dispense Accuracy & Precision Tests – Dispenser #1
20 µL Dispense
Delta ODs @630/750 nm
9
10
11
12
5 µL Dispense
Delta ODs @630/750 nm
Synergy 2 Dispense Accuracy & Precision Tests – Dispenser #2
80 µL Dispense
Delta ODs @405/750 nm
8
A
7
A
B
6
B
C
5
C
D
4
D
E
3
E
F
2
F
G
1
G
g
H
5 µL weight:
H
g
%
%
20 µL weight:
Accuracy % Error:
g
%
80 µL weight:
Accuracy % Error:
Must be <= 20.0%
†F
†F
0.1600 g
†P
Expected weight:
%
Must be <= 5.0%
†F
0.6400 g
†P
Expected weight:
†F
2.5600 g
†P
†P
Expected weight:
Accuracy % Error:
Must be <= 2.0%
Standard Deviation:
Must be <= 10.0%
%CV:
Standard Deviation:
%
Standard Deviation:
†F
Mean:
%CV:
†P
Mean:
%
Must be <= 7.0%
Reviewed/
Approved By:
†F
Tested By:
Signature:
†P
Mean:
%CV:
Must be <= 2.0%
Reader S/N:
Signature:
Reader Model:
Reading Date:
Comments:
Chapter 6
Preventive Maintenance
This chapter provides instructions for maintaining the reader and
dispense module (if used) to ensure that they continue to perform
to specification.
Preventive Maintenance ............................................................138
Warnings and Precautions .........................................................139
Clean Exposed Surfaces............................................................141
Inspect/Clean Excitation and Emission Filters...............................141
Inspect/Clean Mirrors ...............................................................142
Flush/Purge the Fluid Path ........................................................143
Run a Dispense Protocol (Optional) ............................................144
Empty/Clean the Tip Priming Trough ..........................................145
Clean the Priming Plate ............................................................145
Clean the Internal Components..................................................146
138 | Chapter 6: Preventive Maintenance
Preventive Maintenance
A general Preventive Maintenance regimen for all Synergy 2 models includes periodically
cleaning all exposed surfaces and inspecting/cleaning the Emission and Excitation filters
and dichroic mirrors (if equipped).
For models with the external dispense module, additional tasks include flushing/purging
the fluid path, and cleaning the tip prime trough, priming plate, supply bottles, internal
dispense tubing, and injectors.
Daily Cleaning for the Dispense Module
To ensure accurate performance and a long life for the dispense module and injectors,
flush and purge the fluid lines with deionized (DI) water every day or after completing
an assay run, whichever is more frequent. Some reagents may crystallize or harden
after use and clog the fluid passageways. Take special care when using molecules that
are active at very low concentrations (e.g., enzymes, inhibitors). Remove any residual
reagent in the dispense lines using a suitable cleaning solution (review the reagent’s
package insert for specific recommendations).
Flushing the tubing at the end of each day, letting the DI water soak overnight, and
then purging the lines at the beginning of each day ensures optimal performance of the
dispense system. BioTek recommends performing a visual inspection of the dispense
accuracy before running an assay protocol that includes dispense steps.
BioTek also recommends flushing the module with DI water before conducting the
decontamination procedure described in Chapter 7, As Needed Maintenance.
Models with injectors: Accumulated algae, fungi, or mold may require
decontamination. See Chapter 7, As Needed Maintenance for
instructions.
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Warnings and Precautions |
139
Schedule
™ The risk and performance factors associated with your assays may
require performing some or all of the procedures more frequently than
presented in the schedule.
Task
Page
Daily
Quarterly
As Needed
All models:
Clean exposed surfaces
141
Inspect/clean emission and
excitation filters
141
Inspect/clean mirrors
142
Decontamination
162
9
9
annually
before shipment and storage
Models with injectors only:
Flush/purge the fluid path
143
(Optional) Run Dispense protocol
144
Empty/clean tip prime trough
145
Clean priming plate
145
Clean internal components
146
9
9
9
9
9
9
Warnings and Precautions
Read these warnings and precautions before performing maintenance procedures:
Warning! Internal Voltage. Turn off and unplug the instrument
for all maintenance and repair operations.
Important! Do not immerse the instrument, spray it with liquid,
or use a “wet” cloth on it. Do not allow water or other cleaning
solution to run into the interior of the instrument. If this
happens, contact BioTek’s Technical Assistance Center.
Important! Do not apply lubricants to the microplate carrier or
carrier track. Lubricant attracts dust and other particles, which
may obstruct the carrier path and cause errors.
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Warning! Wear protective gloves when handling contaminated
instruments. Gloved hands should be considered contaminated
at all times; keep gloved hands away from eyes, mouth, nose,
and ears.
Warning! Mucous membranes are considered prime entry routes
for infectious agents. Wear eye protection and a surgical mask
when there is a possibility of aerosol contamination. Intact skin is
generally considered an effective barrier against infectious
organisms; however, small abrasions and cuts may not always be
visible. Wear protective gloves when handling contaminated
instruments.
Caution! The buildup of deposits left by the evaporation of
spilled fluids within the read chamber can impact
measurements. Be sure to keep System Test records before and
after maintenance so that changes can be noted.
Caution! Models with injectors. Before removing the reader’s
shroud to expose internal parts, purge the dispense module, turn
off the instrument, and disconnect the fluid line, power cable,
and PC cable.
Warning! The Tungsten lamp assembly is hot when the
instrument is powered on. If the instrument is on, turn it off and
allow the lamp to cool down before attempting to replace it.
Warning! Pinch Hazard. Some areas of the reader can present
pinch hazards when the instrument is operating. These areas are
marked with the symbol shown here. Keep hands/fingers clear
of these areas when the instrument is operating.
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Clean Exposed Surfaces |
141
Clean Exposed Surfaces
Exposed surfaces may be cleaned (not decontaminated) with a cloth moistened (not
soaked) with water or water and a mild detergent. You’ll need:
•
Deionized or distilled water
•
Clean, lint-free cotton cloths
•
Mild detergent (optional)
1. Important: Turn off and unplug the instrument.
2. Moisten a clean cotton cloth with water, or with water and mild detergent. Do not
soak the cloth.
3. Wipe the plate carrier and all exposed surfaces of the instrument.
4. Wipe all exposed surfaces of the dispense module (if used).
5. If detergent was used, wipe all surfaces with a cloth moistened with water.
6. Use a clean, dry cloth to dry all wet surfaces.
™ Models with injectors: If the Tip Priming Trough overflows, wipe the
carrier and the surface beneath the carrier with a dry cotton cloth. If
overflow is significant, you may have to remove the reader’s shroud
and the incubator housing to access the surface beneath the carrier.
See instructions starting on page 146.
Inspect/Clean Excitation and Emission Filters
Laboratory air is used to cool the lamp, and the filters can become dusty as a result. The
filters should be inspected and cleaned at least every three months. You’ll need:
•
Isopropyl, ethyl, or methyl alcohol
•
100% pure cotton balls or high-quality lens-cleaning tissue
•
Cloth gloves
•
Magnifying glass
™ Do not touch the filters with your bare fingers.
1. Turn off and unplug the instrument.
2. Pull down the hinged door on the front of the instrument. Observe the two
thumbscrews within the compartment. The left thumbscrew holds the excitation
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142 | Chapter 6: Preventive Maintenance
(EX) filter wheel in place; the right secures the emission (EM) filter wheel. Remove
each thumbscrew and pull the filter wheel out of the compartment.
3. Inspect the filters for speckled surfaces or a “halo” effect. This may indicate
deterioration due to moisture exposure over a long period of time. If you have any
concerns about the quality of the filters, contact your BioTek representative.
4. Using cotton balls or lens-cleaning tissue moistened with a small amount of highquality alcohol, clean each filter by lightly stroking its surface in one direction.
Ensure that the filters remain in their current locations.
5. Use a magnifying glass to inspect the surface; remove any loose debris from the
cotton ball.
6. Replace the filter wheels in their respective positions and replace the thumbscrews.
Close the hinged door.
Inspect/Clean Mirrors
We recommend inspecting/cleaning the mirrors and polarizing filters (if equipped)
annually, especially if the mirror holder has been handled or changed.
These optical elements are delicate and should be handled as carefully as possible. The
glass and anti-reflective (AR) coated surfaces will be damaged by any contact, especially
by abrasive particles. In most cases, it is best to leave minor debris on the surface.
However, if performance indicators or obvious defects in the mirrors or filters suggest
cleaning them, here are some guidelines:
•
Use of oil-free dry air or nitrogen under moderate pressure is the best method for
removing excessive debris from an optical surface. In the case that the
contamination is not dislodged by the flow of gas, please follow the cleaning
instructions below.
™ All of the mirrors and filters can be easily damaged, especially the
dichroic mirrors. Perform the cleaning steps only when necessary and
always handle the mirror and filters carefully.
•
The purpose of the cleaning solvent is only to dissolve any adhesive contamination
that is holding the debris on the surface. The towel needs to absorb both the
excessive solvent and entrap the debris so that it can be removed from the surface.
Surface coatings on dichroics are typically less hard than the substrate. It is
reasonable to expect that any cleaning will degrade the surface at an atomic level.
Consideration should be given as to whether the contamination in question is more
significant to the application than the damage that may result from cleaning the
surface. In many cases, the AR coatings that are provided to give maximum light
transmission amplify the appearance of contamination on the surface.
BioTek Instruments, Inc.
Flush/Purge the Fluid Path |
143
Materials
•
Cloth gloves
•
Anhydrous reagent-grade ethanol
•
Kimwipes
•
Magnifying glass
•
100% pure cotton balls (for the polarizing filters)
Procedure
1. Turn off the reader and remove its shroud (see page 85 for instructions).
2. Perform the steps in Chapter 4, Filters and Mirrors to remove the mirror holder.
3. Use absorbent towels such as Kimwipes, not lens paper, and wear gloves or use
enough toweling so that solvents do not dissolve oils from your hands which can
seep through the toweling onto the coated surface.
4. Wet the towel with an anhydrous reagent grade ethanol.
5. Drag the trailing edge of the ethanol soaked Kimwipe across the surface of the
component, moving in a single direction. A minimal amount of pressure can be
applied while wiping. However, too much pressure will damage the component.
6. Use the magnifying glass to inspect the surface, if debris is still visible, repeat with a
new Kimwipe.
7. If equipped, clean the polarizing filters. Dampen a cotton ball with alcohol and
gently stroke the surface of the filter to remove dust or fingerprints.
8. Reinstall the mirror holder and replace the shroud.
Flush/Purge the Fluid Path
Applies only to models with injectors.
At the end of each day that the dispense module is in use, flush the fluid path using Gen5’s
priming utility. Leave the fluid to soak overnight or over a weekend, and then purge the
fluid before using the instrument again.
™ This flushing and purging routine is also recommended before
disconnecting the outlet tubes from the back of the reader, and before
decontamination to remove any assay residue prior to applying
isopropyl alcohol or sodium hypochlorite.
To flush the fluid path:
1. Fill two supply bottles with deionized or distilled water. Insert the supply (inlet)
tubes into the bottles.
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144 | Chapter 6: Preventive Maintenance
2. Place the priming plate on the carrier.
3. Select System > Reader Control > Synergy 2 (Com<#>).
4. Click the Dispenser tab and select Dispenser 1.
5. Set the Volume to 5000 µL. Keep the default prime Rate.
6. Click Prime to start the process. When finished, carefully remove the priming plate
from the carrier and empty it.
7. Repeat these steps for Dispenser 2.
Leave the water in the system overnight or until the instrument is used again. Purge
the fluid from the system (see below) and then prime with the dispense reagent
before running an assay.
To purge the fluid from the system:
1. Place the inlet tubes in empty supply bottles or a beaker.
2. Select System > Reader Control > Synergy 2 (Com<#>).
3. Click the Dispenser tab and select Dispenser 1.
4. Set the Volume to 2000 µL.
5. Click Purge to start the process. When finished, repeat these steps for Dispenser 2.
™ After purging the system, you may wish to run a quick Dispense
protocol to visually verify the dispense accuracy.
Run a Dispense Protocol (Optional)
Applies only to models with injectors.
After flushing/purging the system and before running an assay that requires dispense,
take a moment to visually inspect the dispensing accuracy.
1. Create a new protocol in Gen5. Set the Plate Type to match the plate you will use.
2. Add a Dispense step with the following parameters:
•
Select Dispenser 1
•
Set Tip Priming to ‘Before this dispense step’ and Volume to 10 µL.
•
Set the Dispense Volume to 100 µL (or an amount to match your assay
protocol).
•
Adjust the Rate to support the dispensing volume.
2. Add another Dispense step with the same parameters, selecting Dispenser 2.
3. Add a Read step with the following parameters (Gen5 requires a Read step in a
Dispense protocol):
BioTek Instruments, Inc.
Empty/Clean the Tip Priming Trough |
145
•
Select any Detection Method
•
Set the Read Type to Endpoint
•
Click the Full Plate button and de-select the Use All Wells checkbox. This
action leaves only well A1 selected for the Read.
•
Select any wavelength or define one Filter Set
3. Save the protocol using an identifying name, such as “Dispense Observation.”
4. Fill the supply bottles with a DI H2O-Tween solution (e.g., add 1 mL Tween® 20 to
1000 mL of deionized water).
5. Create and run experiment based on the Dispense protocol.
6. Visually assess the fluid level in the wells for accuracy. If the well volume appears to
be unevenly distributed, clean the internal dispense tubes and injector heads as
described in Cleaning the Internal Components starting on page 146.
Empty/Clean the Tip Priming Trough
Applies only to models with injectors.
The tip priming trough is a removable cup located in the left rear of the microplate carrier,
used for performing the Tip Prime. The trough holds approximately 1.5 mL of liquid and
must be periodically emptied and cleaned by the user. Gen5 will instruct you to do this at
the start of an experiment that requires dispensing.
1. Extend the microplate carrier and carefully remove the tip priming trough from its
pocket in the left rear of the carrier.
2. Wash the trough in hot, soapy water. Use a small brush to clean in the corners.
3. Rinse the trough thoroughly and allow it to dry completely.
4. Replace the trough in the microplate carrier.
Clean the Priming Plate
Applies only to models with injectors.
Clean the priming plate regularly to prevent bacteria growth and residue buildup. Wash
the plate in hot soapy water, using a small brush to clean in the corners. Rinse thoroughly
and allow it to dry completely.
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146 | Chapter 6: Preventive Maintenance
Clean the Internal Components
Applies only to models with injectors.
For models without injectors, the internal chamber and probes are not
customer-accessible. Contact BioTek’s Technical Assistance Center with
any questions about your particular model.
The reader’s internal dispense tubes and injector heads require routine cleaning at least
quarterly and possibly more frequently depending on the type of fluids dispensed.
Cleaning inside the reader is required when fluid has spilled inside the instrument and/or
if an unusually high background signal has been flagged by the assay controls.
Start with Remove the Reader’s Shroud and execute the procedures that meet your needs,
in the order in which they are presented. Finish with Reassemble the Components.
The buildup of deposits left by the evaporation of spilled fluids within
the read chamber can impact performance of the fluorescence,
luminescence, and absorbance functions. Perform a System Test before
and after maintenance so that any changes in performance can be noted.
Wear protective gloves and safety glasses when performing the
procedures.
Required Materials
•
For all tasks, protective gloves and safety glasses
•
For removing the shroud, Phillips screwdriver
•
For cleaning the internal dispense tubes and injector heads, and for wiping the
surface under the plate carrier:
¾
Mild detergent or isopropyl alcohol
¾
Clean, lint-free cotton cloths
¾
Deionized or distilled water
¾
Stylus (stored in a cylinder affixed to the back of the dispense module or
reader; BioTek PN 2872304)
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147
Remove the Reader’s Shroud
Before removing the shroud, purge the dispense module (see page 143),
and then turn off and disconnect the reader from its power supply, the
PC, and the dispense module.
1. Purge the dispense module of fluid.
2. Disconnect power and all cables. Set the external dispense module aside.
3. Clear the work surface around the reader so you can easily access all sides of the
instrument.
4. Remove two screws: one on each side of reader at the lower-rear corner.
Remove two side
screws (one on each
side of the reader)
Figure 40: Screws in the lower-rear corners
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148 | Chapter 6: Preventive Maintenance
5. Stand facing the front of the instrument. Grasp both sides of the shroud, slide it
toward you, and pull it straight off the instrument. Set the shroud aside.
Figure 41: Removing the reader’s shroud
™ To reinstall the shroud, rest its bottom on the table in front of the
reader and gently slide it into place. Internal wheels roll along the
reader’s bottom track to properly reposition the shroud.
Clean the shroud’s air filters
If dust accumulates on the shroud’s air filters:
•
Use a vacuum cleaner to clean the filters in place, or
•
Remove the grate holding the filters and soak the filters in mildly soapy
water. Rinse well and reinstall.
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Remove the Internal Dispense Tubes and Injector Heads
1. Locate the tubing ports on the reader’s back wall. Turn each tube’s thumbscrew
counterclockwise and gently pull the tube from the port (Figure 43).
2. Locate the injector heads. Turn each tube’s thumbscrew counterclockwise and gently
pull the tube from its injector tip (Figure 44).
3. Turn each injector head counterclockwise and gently pull it out of the socket (Figure
45).
Tubing ports (2) for connecting
the two internal dispense tubes to
the two external dispense tubes
Internal tubes
(2, black) for
delivering fluid
to the injector
heads
Injector heads
(2, white) for dispensing
fluid into microwells
Figure 42: Internal components for the injector system
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150 | Chapter 6: Preventive Maintenance
Figure 43: Disconnect the dispense tubes from the back wall
Figure 44: Disconnect the dispense tubes from the injector heads
BioTek Instruments, Inc.
Clean the Internal Components |
Figure 45: Remove the injector heads
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152 | Chapter 6: Preventive Maintenance
Clean the Dispense Tubes and Injector Heads
As discussed on page
138, some reagents
can crystallize and
clog the tubing and
injector heads.
Daily flushing and
purging can help to
prevent this, but
more rigorous
cleaning may be
necessary if reagent
has been allowed to
dry in the tubing
and/or injectors.
Figure 46: Injector heads and internal dispense tubes
To clean the internal tubes, soak them in hot, soapy water to soften and dissolve any
hardened particles. Flush each tube by holding it vertically under a stream of water.
To clean the injector tips:
•
Gently insert the stylus into each injector tip to clear any blockages. The stylus
(BioTek PN 2872304) is stored in a cylinder affixed to the back of the dispense
module or reader.
•
Stream water through the pipe to be sure it is clean. If the water does not
stream out, try soaking in hot, soapy water and then reinserting the stylus.
™ Do not bend the injector tips. A bent tip might not dispense accurately.
™ Do not remove the o-rings (if equipped).
O-ring; do
not remove
Figure 47: Injector head and stylus
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153
Clean Inside the Reader
The internal surface and some components should be cleaned if reagent has spilled or
if an unusually high background signal has been identified.
•
BioTek recommends performing this process in conjunction with the previously
defined steps for cleaning the dispense tubes and injectors.
•
For this procedure you will need:
¾
Deionized or distilled water and mild detergent (optional)
¾
Two or more lint-free cotton cloths
Knob for raising
or lowering the
optics armature
Heater wires:
2 must be
disconnected
Three black knobs
hold the incubator
housing in place
Incubator housing
Figure 48: Internal components discussed in this section
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154 | Chapter 6: Preventive Maintenance
Remove the Incubator Housing
1. If you have not already done so, unplug the reader and remove its shroud (page
147).
2. Disconnect two heater wires located on the reader’s back wall. The two wires are in
positions 1 and 3 of the top row (see below). To disconnect a wire, depress the tab
and slide it off the board.
c
d
e f
Depress tab to
disconnect wire
Figure 49: Disconnect heater wires 1 and 3 in the top row
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155
3. Locate and remove the three knobs that secure the incubator housing.
Figure 50: Removing the knobs that secure the incubator housing
4. Turn the optic arm knob clockwise to raise the optics armature as high as it will go.
Figure 51: Raising the optics armature
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156 | Chapter 6: Preventive Maintenance
5. Lift the left side of the incubator housing and carefully slide it out.
6. Turn over the incubator housing and clean the surface with water and mild
detergent. Set it aside and let it dry completely.
™ When replacing the incubator housing, the two “forks” on its right side
should wrap around the holding screws. The forks should not slide
under the fixed foam.
Figure 52: Removing the incubator housing
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Clean the Internal Components |
157
Clean the Reader’s Surface
1. After you remove the incubator housing, slide the microplate carrier to the left to
engage the support pin and then toward the front of the reader.
Slide open the
microplate carrier
Figure 53: Cleaning the inside of the reader
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158 | Chapter 6: Preventive Maintenance
2. Moisten (do not soak) a clean cotton cloth with water, or with water and mild
detergent, or alcohol. Wipe all sides of the plate carrier. Wipe the instrument’s
horizontal surface.
If the injector heads were removed from the reader, use the cloth to gently clean
the underside of the optical armature around the injector head holders.
™ Do not apply pressure. Components are easily damaged.
3. If detergent was used, wipe the surfaces with a cloth moistened with water.
4. Use a clean, dry, lint-free cloth to dry all wet surfaces.
Reassemble the Components
1. Slide the microplate carrier all the way into the instrument.
2. If the incubator housing was removed, reinstall it.
•
Observe the two forks on the right side of the housing; they must wrap
around the screws on either side of the optics armature. Do not slide the
forks under the foam.
Figure 54: Incubator housing “fork” in the correct position
•
Secure the incubator housing with the three knobs (Figure 50).
•
Reconnect the two heater wires to the rear wall of the reader: the white wire
fits into position 1 (Figure 49) in the top row of wires; the black wire fits
into position 3.
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3. Insert the two injector heads into their sockets. Ensure that they are properly seated;
the knurled plastic should sit flush against the surface, as shown below.
Figure 55: Injector head properly seated
4. Attach the two internal dispense tubes to the injector heads (Figure 44). Do not
overtighten the thumbscrews!
5. Attach the other end of the two dispense tubes to the tubing ports on the rear wall of
the reader. They can go into either port.
6. Review the steps you just performed to make sure the components have been
properly reassembled.
7. Slide the shroud onto the instrument, page 147.
8. Replace the two side screws to secure the shroud to the base.
Verify Performance
After reassembling the instrument, verify that the instrument is functioning properly:
•
Connect power to the reader and turn it on. Allow the startup system test to
complete. Run a System Test using Gen5; all tests should pass.
•
Run any required OQ/PQ tests.
Synergy 2 Operator’s Manual
160 | Chapter 6: Preventive Maintenance
BioTek Instruments, Inc.
Chapter 7
As Needed Maintenance
This chapter contains maintenance and component-replacement
procedures that need to be performed only occasionally.
Decontamination .....................................................................162
Procedure for Models without Injectors .......................................164
Procedure for Models with Injectors............................................165
Replace the Tungsten Lamp ......................................................169
Replace a Syringe ....................................................................171
162 | Chapter 7: As Needed Maintenance
Decontamination
Any laboratory instrument that has been used for research or clinical analysis is
considered a biohazard and requires decontamination prior to handling.
Decontamination minimizes the risk to all who come into contact with the instrument
during shipping, handling, and servicing. Decontamination is required by the U.S.
Department of Transportation regulations.
Persons performing the decontamination process must be familiar with the basic setup and
operation of the instrument.
BioTek Instruments, Inc., recommends the use of the following
decontamination solutions and methods based on our knowledge of the
instrument and recommendations of the Centers for Disease Control and
Prevention (CDC). Neither BioTek nor the CDC assumes any liability for
the adequacy of these solutions and methods. Each laboratory must
ensure that decontamination procedures are adequate for the
biohazard(s) they handle.
Wear prophylactic gloves when handling contaminated instruments.
Gloved hands should be considered contaminated at all times; keep
gloved hands away from eyes, mouth, and nose. Eating and drinking
while decontaminating instruments is not advised.
Mucous membranes are considered prime entry routes for infectious
agents. Wear eye protection and a surgical mask when there is a
possibility of aerosol contamination. Intact skin is generally considered
an effective barrier against infectious organisms; however, small
abrasions and cuts may not always be visible. Wear protective gloves
when performing the decontamination procedure.
BioTek Instruments, Inc.
Decontamination |
Required Materials
For all models:
•
Sodium hypochlorite (NaClO, or bleach)
•
70% isopropyl alcohol (as an alternative to bleach)
•
Deionized or distilled water
•
Safety glasses
•
Surgical mask
•
Protective gloves
•
Lab coat
•
Biohazard trash bags
•
125 mL beakers
•
Clean, lint-free cotton cloths
Additional materials for models with injectors:
•
Phillips screwdriver
•
Small brush
•
(Optional) Mild detergent
Synergy 2 Operator’s Manual
163
164 | Chapter 7: As Needed Maintenance
Procedure for Models without Injectors
The sodium hypochlorite (bleach) solution is caustic; wear gloves and
eye protection when handling the solution.
Turn off and unplug the instrument for all decontamination and cleaning
procedures.
Do not immerse the instrument, spray it with liquid, or use a “wet”
cloth. Do not allow the cleaning solution to run into the interior of the
instrument. If this happens, contact the BioTek Service Department.
1. Turn off and unplug the instrument.
2. Prepare an aqueous solution of 0.5% sodium hypochlorite (bleach). If the effects of
bleach are a concern, 70% isopropyl alcohol may be used.
™ Check the percent NaClO of the bleach you are using. Commercial
bleach is typically 10.0% NaClO; prepare a 1:20 dilution. Household
bleach is typically 5.0% NaClO; prepare a 1:10 dilution.
3. Moisten a cloth with the bleach solution or alcohol. Do not soak the cloth.
4. Open the plate carrier access door and slide out the carrier.
5. Wipe the plate carrier and all exposed surfaces of the instrument.
6. Wait 20 minutes. Moisten a cloth with deionized (DI) or distilled water and wipe all
surfaces of the instrument that have been cleaned with the bleach solution or alcohol.
7. Use a clean, dry cloth to dry all wet surfaces.
8. Reassemble the instrument as necessary.
9. Discard the used gloves and cloths using a Biohazard trash bag and an approved
Biohazard container.
BioTek Instruments, Inc.
Procedure for Models with Injectors |
165
Procedure for Models with Injectors
™ Perform the Routine Procedure when the reader is functioning
normally. If you are unable to perform a prime due to a system failure,
perform the Alternate Procedure described on page 167.
Routine Procedure
If disinfecting with sodium hypochlorite (bleach), be sure to
flush repeatedly with deionized water to remove the bleach.
After disinfecting with sodium hypochlorite, perform the rinse
procedure provided on page 166.
If disinfecting with alcohol, do not immediately prime with
deionized water, because the drying effect of the alcohol is an
important aspect of its disinfectant properties.
Clean Exposed Surfaces
1. Turn off and unplug the instrument.
2. Prepare an aqueous solution of 0.5% sodium hypochlorite (bleach). If the effects
of bleach are a concern, 70% isopropyl alcohol may be used.
™ Check the percent NaClO of the bleach you are using. Commercial
bleach is typically 10.0% NaClO; prepare a 1:20 dilution.
Household bleach is typically 5.0% NaClO; prepare a 1:10 dilution.
3. Open the plate carrier access door and slide out the carrier.
4. Moisten a cloth with the bleach solution or alcohol. Do not soak the cloth.
5. Wipe the plate carrier and the exposed surfaces of the external dispense module.
6. Wait 20 minutes. Moisten a cloth with deionized (DI) or distilled water and wipe
all surfaces that have been cleaned with the bleach solution or alcohol.
7. Use a clean, dry cloth to dry all wet surfaces.
8. Reassemble the instrument as necessary.
9. If the dispense module is installed, purge any fluid (see Flush/Purge the Fluid
Path in Chapter 6) and detach the outlet tubes from the back of the instrument. If
it is not installed, attach only the dispense module’s communication cable to the
instrument. Remove the supply bottles and their holders.
10. Perform the decontamination procedures described below.
Synergy 2 Operator’s Manual
166 | Chapter 7: As Needed Maintenance
Decontaminate the Fluid Lines
1. Place a beaker with 20 mL of 0.5% sodium hypochlorite solution or 70%
isopropyl alcohol near SYRINGE 1 on the dispense module.
2. Place the SYRINGE 1 inlet tube in the beaker.
3. If you have not already done so, detach the dispense module’s outlet tubes from
the instrument’s rear panel. Place the ends of the outlet tubes in an empty beaker
and set the beaker next to the dispense module.
4. Launch Gen5, select System > Reader Control, and click the Dispenser tab.
5. Select Dispenser 1, enter a Volume of 5000 µL, and keep the default dispense
Rate.
6. Place the priming plate on the carrier.
7. Run two prime cycles, for a total of 10,000 µL.
8. Wait at least 20 minutes to allow the solution to disinfect the tubing.
9. Remove the inlet tube from the beaker of disinfectant solution.
10. From the Reader Control dialog, change the Volume to 1000 µL.
11. Run one prime cycle, to flush the disinfectant out of the fluid lines.
12. Empty the beaker containing the outlet tubes. Put the tubes back in the empty
beaker.
13. If sodium hypochlorite (bleach) was used, perform Rinse the Fluid Lines below.
Otherwise, (or after performing the Rinse procedure), repeat steps 1–13 for
SYRINGE 2/Dispenser 2.
Rinse the Fluid Lines
™ Perform this procedure only if decontamination was performed using
sodium hypochlorite.
1. Place a beaker containing at least 30 mL of deionized water on the dispense
module.
2. Place the SYRINGE 1 or 2 inlet tube in the beaker.
3. If you have not already done so, place the outlet tubes in an empty beaker.
4. From the Reader Control dialog, select Dispenser 1 or 2, set the Volume to 5000
µL, and keep the default dispense Rate.
5. Run five prime cycles, for a total of 25,000 µL.
6. Pause for 10 minutes and then run one prime cycle with 5000 µL. This will allow
any residual sodium hypochlorite to diffuse into the solution and be flushed out
with the next prime.
BioTek Instruments, Inc.
Procedure for Models with Injectors |
167
7. Empty the beaker containing the outlet tubes.
8. Wipe all surfaces with deionized water.
9. Discard the used gloves and cloths using a biohazard trash bag and an approved
biohazard container.
Clean the Internal Tubing and Injectors
Perform the procedures under “Clean the Internal Components” in Chapter 6,
Preventive Maintenance.
Decontaminate the Tip Priming Trough and Priming Plate
1. Remove the tip priming trough from the instrument’s microplate carrier.
2. Wash the tip priming trough and priming plate in hot, soapy water. Use a small
brush or cloth to clean the corners of the trough and plate.
3. To decontaminate, soak the trough and plate in a container of 0.5% sodium
hypochlorite or 70% isopropyl alcohol for at least 20 minutes.
•
If decontaminating in a bleach solution, thoroughly rinse the trough and
plate with DI water.
•
If decontaminating with alcohol, let the trough and plate air dry.
4. Discard the used gloves and cloths using a biohazard trash bag and an approved
biohazard container.
Alternate Procedure
If you are unable to prime the reader due to a system failure, decontaminate the
instrument and the dispense module as follows:
1. Perform the procedures under “Clean the Internal Components” in Chapter 6,
Preventive Maintenance. When finished, leave the shroud off the reader and
proceed to step 2.
2. Prepare an aqueous solution of 0.5% sodium hypochlorite (bleach). If the effects
of bleach are a concern, 70% isopropyl alcohol may be used.
™ Check the percent NaClO of the bleach you are using. Commercial
bleach is typically 10.0% NaClO; prepare a 1:20 dilution.
Household bleach is typically 5.0% NaClO; prepare a 1:10 dilution.
3. Slide the microplate carrier out of the instrument.
4. Moisten a cloth with the bleach solution or alcohol. Do not soak the cloth.
5. Use the cloth to wipe:
•
All surfaces of the shroud
•
All surfaces of the plate carrier
Synergy 2 Operator’s Manual
168 | Chapter 7: As Needed Maintenance
•
The instrument’s rear panel
•
The exposed surfaces of the dispense module, including the syringe
valves
6. Remove the external tubing and the syringes from the dispense module and soak
them in the bleach or alcohol solution. Wait for 20 minutes.
7. Moisten a cloth with DI or distilled water and wipe all surfaces that have been
cleaned with the bleach solution or alcohol.
8. Rinse all tubing and the syringes with DI water.
9. Use a clean, dry cloth to dry all surfaces on the instrument and the dispense
module.
10. Reassemble the instrument and dispense module as necessary.
11. Discard the used gloves and cloths using a biohazard trash bag and an approved
biohazard container.
BioTek Instruments, Inc.
Replace the Tungsten Lamp |
169
Replace the Tungsten Lamp
The tungsten bulb is expected to operate without replacement for a minimum of 1000
hours. The intensity of the bulb will slowly drop over time until the reader’s System Test
detects a low signal and displays an error message. In addition, error code 2901 may be
displayed during normal operation. The lamp should be replaced at this time; contact
BioTek and order part number 7080500.
When the reader’s front panel is opened, the tungsten lamp is located behind a lightblocking panel with the hot surface warning label. The lamp is secured by a bracket that
also holds a condenser lens and a heat absorber. Two cables extend from the back of the
lamp to plug into the reader.
The lamp is hot when the instrument is on. Before replacing the lamp,
turn off the reader and allow the lamp to cool for at least 15 minutes.
Do not touch the glass lenses! Fingerprints on the condenser lens or heat
absorber may negative affect performance.
1. Turn off and unplug the reader. Wait at least 15 minutes for the lamp to cool.
2. Remove the reader’s shroud (see page 147).
3. Remove the EX filter wheel and set aside.
4. Grasp the light-blocking panel and slide the assembly toward you, out of the reader.
Cables
Lightblocking
panel
Figure 56: Light panel and EX wheel removed for lamp replacement
Synergy 2 Operator’s Manual
170 | Chapter 7: As Needed Maintenance
5. Unplug the lamp’s cables from the reader.
6. Gently, without touching any glass components, unscrew one of the thumbscrews
holding the bulb in its wire bracket.
7. When the bracket is free, remove the old bulb.
Thumbscrews
Figure 57: Lamp replacement
8. Insert the new bulb, position the wire bracket, and tighten the thumbscrew.
9. Align the lamp assembly with the reader, preparing to slide its top metal plate into
its slot, and plug in the red cables. The cables can go into either plug.
10. Slide the lamp assembly into place without touching any other internal component.
You may need to move the cables slightly downward to make room for the lamp.
11. Reinstall the EX filter wheel and the reader’s shroud.
12. Plug in and turn on the reader. If the system test passes the lamp replacement was
successful. Otherwise, note any errors and consult Appendix B, Error Codes.
BioTek Instruments, Inc.
Replace a Syringe |
171
Replace a Syringe
Refer to Chapter 6, Preventive Maintenance for cleaning procedures you should perform
regularly and also in the case of poor performance. If cleaning the dispense module does
not eliminate performance problems, or if a syringe is leaking, perform this procedure to
replace a faulty syringe. Contact BioTek to order replacement syringes.
1. In Gen5, select System > Reader Control > Synergy 2 (Com<#>) and click
the Dispenser tab.
2. Select the appropriate Dispenser number (1 or 2) associated with the syringe and
click Maintenance. The syringe will move to its furthest-from-home position.
3. Using your fingers, remove the bottom thumbscrew that secures the syringe,
underneath the bracket.
4. Turn the top thumbscrew to disengage the syringe from the valve.
5. Remove the new syringe from its protective box. (The syringe should be assembled
in one piece; if not, see “Install the Dispense Module” in Chapter 2, Installation.)
6. Hold the syringe vertically with the threaded end at the top. Screw the top of the
syringe into the bottom of the syringe valve. Finger-tighten only.
7. Pull down the bottom of the syringe until it rests inside the hole in the bracket.
8. Pass a thumbscrew up through the hole and thread it into the bottom of the syringe.
Hold the syringe from rotating while tightening the screw. Finger-tighten only.
9. In Gen5, select System > Reader Control > Synergy 2 (Com<#>). Click the
Dispenser tab and click Initialize.
Top
thumbscrews
Syringes
Syringe
brackets
Bottom
thumbscrews
Figure 58: Dispense module syringes
Synergy 2 Operator’s Manual
172 | Chapter 7: As Needed Maintenance
BioTek Instruments, Inc.
Appendix A
Specifications
This chapter contains BioTek’s published specifications for the
Synergy 2.
General Specifications ..............................................................174
Absorbance Specifications .........................................................175
Luminescence Specifications......................................................176
Fluorescence Specifications .......................................................176
Models with Injectors ...............................................................179
174 | Appendix A: Specifications
General Specifications
Microplates
The Synergy 2 accommodates standard 6-, 12-, 24-, 48-, 96-, 384-, and 1536-well
microplates with 128 x 86 mm geometry, and the BioTek Take3 Multi-Volume Plate.
Note: 1536-well microplates are not supported for luminescence measurements.
Maximum Plate Height:
Absorbance mode: plates up to 0.8” (20.30 mm) high
Fluorescence/Luminescence modes: plates up to 1.25” (31.75 mm) high
PCR tube trays up to 1.25” (31.75 mm) high (may require an adapter)
Injector models dispense to standard height 6-, 12-, 24-, 48-, and 96-well microplates.
Hardware & Environmental
Light Source:
Absorbance: Xenon flash light source, 10W maximum average
power, lamp life 1 billion flashes (not user-changeable)
Fluorescence: Tungsten quartz halogen, 20W power, lamp life
1000 hours (user-changeable)
TRF (and optionally for FI and FP): Xenon flash light source,
60W maximum average power, lamp life 1 billion flashes (not
user-changeable)
Dimensions:
17.5” D x 15” W x 17.5” H
44.5 cm D x 38.5 cm W x 44.5 cm H
Weight:
With all modules installed, without power supply or dispense
module attached, approx. 57 lbs. (25.8 kg)
Environment:
Operational temperature 18º to 40°C
Humidity:
10% to 85% relative humidity (non-condensing)
Power Supply:
24-volt external power supply compatible with
100-240 V~; +10% @50-60 Hz
Power Consumption:
250W maximum
Incubation:
Temperature control range from 4º over ambient to 50ºC.
Temperature variation ± 0.50°C across the plate @ 37°C (250
µl per well with the plate sealed).
Plate Shaking:
Low, Medium, High and Variable shaking speeds.
Shake time is programmable by the user.
BioTek Instruments, Inc.
Absorbance Specifications |
Absorbance Specifications
Accuracy, Linearity, Repeatability
All qualifications were conducted using 96-/384-well, flat bottom microplates.
For the performance described here, the Gain on the Optics Test should be
below 10.0.
Measurement Range:
0.000 to 4.000 OD
Accuracy:
0.000 to 2.000
0.000 to 2.000
2.000 to 2.500
2.500 to 3.000
0.000 to 1.000
OD
OD
OD
OD
OD
±1.0%
±2.0%
±3.0%
±3.0%
±1.0%
±0.010
±0.010
±0.010
±0.010
±0.010
Linearity:
0.000 to 2.000
0.000 to 2.000
2.000 to 2.500
2.500 to 3.000
0.000 to 1.000
OD
OD
OD
OD
OD
±1.0%
±2.0%
±3.0%
±3.0%
±1.0%
Normal/Rapid modes, 96-well plates
Normal/Rapid modes, 384-well plates
Normal/Rapid modes, 96-/384-well plates
Normal mode, 96-well plates
Sweep mode, 96-/384-well plates
Repeatability:
0.000 to 2.000
2.000 to 2.500
2.500 to 3.000
0.000 to 1.000
OD
OD
OD
OD
±1.0%
±3.0%
±3.0%
±2.0%
±0.005
±0.005
±0.005
±0.010
OD
OD
OD
OD
OD
OD
OD
OD
OD
Normal/Rapid modes, 96-well plates
Normal/Rapid modes, 384-well plates
Normal/Rapid modes, 96-/384-well plates
Normal mode, 96-well plates
Sweep mode, 96-/384-well plates
Normal/Rapid modes, 96-/384-well plates
Normal/Rapid modes, 96-/384-well plates
Normal mode, 96-/384-well plates
Sweep mode, 96-/384-well plates
Optics
λ range:
200 to 999 nm
λ accuracy:
± 2 nm
λ repeatability:
± 0.2 nm
λ bandpass:
2.4 nm
Detector:
Photodiodes (2)
Measurements are reference channel-corrected for light
source fluctuation
Synergy 2 Operator’s Manual
175
176 | Appendix A: Specifications
Read Timing
Endpoint read time is from plate start to plate stop.
Kinetic read time is from A1 to A1 read positions.
Endpoint
96-well plate
Endpoint
384-well plate
Endpoint
1536-well plate
Kinetic
96-well
Kinetic
384-well
Kinetic
1536-well
Normal 0 ms delay:
Normal 100 ms delay:
Sweep:
Single 630 nm
34 sec
43 sec
22 sec
Dual 630/450 nm
53 sec
73 sec
35 sec
Normal 0 ms delay:
Normal 100 ms delay:
Sweep:
Single 630 nm
77 sec
115 sec
32 sec
Dual 630/450 nm
143 sec
220 sec
56 sec
Normal 0 ms delay:
Normal 100 ms delay:
Sweep:
Single 630 nm
231 sec
385 sec
53 sec
Dual 630/450 nm
450 sec
757 sec
97 sec
Normal 0 ms delay:
Normal 100 ms delay:
Sweep:
Single 630 nm
21 sec
31 sec
11 sec
Normal 0 ms delay:
Normal 100 ms delay:
Sweep:
Single 630 nm
66 sec
104 sec
22 sec
Normal 0 ms delay:
Normal 100 ms delay:
Sweep:
Single 630 nm
219 sec
373 sec
42 sec
Luminescence Specifications
50 amol/well flash ATP in a 96-well plate (low-noise #49984 PMT)
500 amol/well flash ATP in a 96-well plate (red-shifted #49721 PMT)
BioTek Instruments, Inc.
Fluorescence Specifications |
177
Fluorescence Specifications
Read Timing
Because of the possible wide variations in setup, the following benchmark conditions are
specified:
Excitation Filter:
485/20 nm
Emission Filter:
528/20 nm
Samples per well:
10
Delay before sampling:
100 ms
Delay between samples:
1 ms
96-well read:
99 sec
384-well read:
339 sec
1536-well read:
1256 sec
Optical Probes
Bottom position: 1.5, 3, or 5 mm diameter probes can be installed
Top position: 3 mm diameter fixed, with motor-driven moveable apertures to reduce the
diameter to support different plate formats
Sensitivity
The following specifications apply to 96-well read formats using the Tungsten bulb.
5 mm optical probe
Bottom reading
10 pg/mL solution of Sodium Fluoroscein in PBS
40 reads per location averaged, 350 ms delay before read
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 528/20
Hellma 96-well quartz plate or Greiner SensoPlate
62.5 ng/mL solution of Propidium Iodide in PBS
40 reads per location averaged, 350 ms delay before read
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 645/40
Corning Costar 96-well plate with black sides, clear bottom
3 mm optical probe
Bottom reading
20 pg/mL solution of Sodium Fluoroscein in PBS
40 reads per location averaged, 350 ms delay before read
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 528/20
Hellma 96-well quartz plate
3 mm optical probe
Bottom reading
125 ng/mL solution of Propidium Iodide in PBS
40 reads per location averaged, 350 ms delay before read
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 645/40
Corning Costar 96-well plate with black sides, clear bottom
Synergy 2 Operator’s Manual
178 | Appendix A: Specifications
Sensitivity
The following specifications apply to 96-well read formats using the Tungsten bulb.
1.5 mm optical
probe Bottom
reading
40 pg/mL solution of Sodium Fluoroscein in PBS
40 reads per location averaged, 350 ms delay before read
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 528/20
Hellma 96-well quartz plate
250 ng/mL solution of Propidium Iodide in PBS
40 reads per location averaged, 350 ms delay before read
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 645/40
Corning Costar 96-well plate with black sides, clear bottom
3 mm fixed optical
probe with movable
apertures, Top
reading
2 pg/mL solution of Sodium Fluoroscein in PBS
40 reads per location averaged, 5 mm Z-axis offset, 350 ms
delay before read
200 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 528/20, Dichroic 510 nm
Corning Costar 96-well solid black plate
0.16 ng/mL solution of Methylumbelliferone in CBB
40 reads per location averaged, 5 mm Z-axis offset, 350 ms
delay before read
150 µL per well signal-to-noise ratio greater than 2
Excitation 360/40, Emission 460/40, 50% mirror
Corning Costar black strips
Time-Resolved Fluorescence
96/384-well plates:
250 fM Europium (plate and well modes), 20 reads per
location, 5 mm Z-axis offset, 350 ms delay before read
Integration Interval:
20 to 16000 µs
Delay:
0 to 16000 µs
Granularity:
1-µs steps
Fluorescence Polarization
96/384-well plates:
5 mP at 1 nM Sodium Fluorescein, 60 reads per location,
5 mm Z-axis offset 350 ms delay before read
BioTek Instruments, Inc.
Models with Injectors |
179
Models with Injectors
Dispense/Read
Specifications apply to models with the dual-reagent dispense module
Plate Type:
Dispenses to standard 6-, 12-, 24-, 48-, and 96-well microplates
with standard 128 x 86 mm geometry
Detection Method:
Absorbance, Fluorescence, Luminescence
Volume Range:
5-1000 µL with a 5-20 µL tip prime
Accuracy:
Dispensing deionized water with 0.1% Tween 20
at room temperature:
± 1 µL at 5-50 µL
± 2% at 51-1000 µL
Precision:
Dispensing a 200 µL solution of deionized water,
0.1% Tween 20, and dye at room temperature:
< 2.0% for volumes of 50-200 µL
< 4.0% for volumes of 25-49 µL
< 7.0% for volumes of 10-24 µL
< 10.0% for volumes of 5-9 µL
Synergy 2 Operator’s Manual
180 | Appendix A: Specifications
BioTek Instruments, Inc.
Appendix B
Error Codes
This chapter contains the most common and easily resolved
Synergy 2 error codes that may appear in Gen5.
Overview ................................................................................182
Error Codes ............................................................................183
182 | Appendix B: Error Codes
Overview
When a problem occurs during operation with the Synergy 2, an error code will appear in
Gen5. Error codes typically contain four characters, such as “2B0A,” and in most cases are
accompanied by descriptive text, such as “Priming plate not detected.” With many errors,
the instrument will beep repeatedly; press the carrier eject button to stop this alarm.
Some problems can be solved easily, as with 2B0A (place a priming plate on the carrier).
Some problems can be solved only by trained BioTek Service personnel. This appendix
lists the most common and easily resolved error codes that you may encounter.
™ Error codes beginning with “A” (e.g., A100) indicate conditions that
require immediate attention. If this type of code appears, turn the
instrument off and on. If the System Test does not conclude
successfully, record the error code and contact BioTek’s Technical
Assistance Center.
If an error code appears in Gen5, you may want to run a System Test for diagnostic
purposes. In Gen5, select System > Diagnostics > Run System Test.
If an error message appears while an experiment is in process and after
having received measurement data, it is your responsibility to determine
if the data is valid.
Contact Info: BioTek Service/TAC
Use this appendix to diagnose problems and solve them if possible. If you need further
assistance, contact BioTek’s Technical Assistance Center.
Phone:
800-242-4685 (toll free in the U.S.)
802-655-4740 (outside the U.S.)
Fax:
802-654-0638
E-Mail:
tac@biotek.com
™ For errors that are displayed during operation of the reader with the
BioStack Microplate Stacker, refer to the BioStack Operator’s Manual.
BioTek Instruments, Inc.
Error Codes |
183
Error Codes
This table lists the most common and easily resolved error codes that you may encounter.
If an error code appears in Gen5, look for it here. If you find the code, follow the
suggestions provided for solving the problem. If you cannot find the code or if you are
unable to solve the problem, please contact BioTek’s Technical Assistance Center. The
Gen5 Help system also provides troubleshooting tips.
Code
Description and Possible Remedies
0202
0203
EX/EM filter wheel (respectively) did not home
Generally, this error indicates the Excitation or Emission filter wheel is not
seated properly in the reader. Remove the wheel, ensure each filter or plug
is properly positioned, and reinstall it securely. Restart the reader.
020B
020C
Dispenser syringe 1 or 2 (respectively) did not home
Generally, this error indicates the syringe was not properly installed. Make
sure the syringe’s thumbscrews are properly threaded. (Refer to the
Installation chapter for instructions.) Restart the reader.
0402
0403
EX/EM filter wheel (respectively) failed positional verify
Generally, this error indicates the Excitation or Emission filter wheel is not
seated properly in the reader. Remove the wheel, ensure each filter or plug
is properly positioned, and reinstall it securely. Restart the reader.
040B
040C
Dispenser syringe 1 or 2 failed position verify
Generally, this error indicates the syringe was not properly installed. Make
sure the syringe’s thumbscrews are properly threaded. (Refer to the
Installation chapter for instructions.) Restart the reader.
050x
Light beam saturated (too much light). Relative Fluorescing Units (RFU)
reached (99999).
This error can indicate one of several scenarios. It is possibly due to incorrect
chemistry, e.g., the fluorescence standards dispensed to the plate exceed
expectations.
See also the description for 0E01.
Verify that the microplate carrier access door is closing properly and no
ambient light is getting into the read chamber.
If you are manually setting the Sensitivity values in your Gen5 procedure, try
using the Automatic Sensitivity Adjustment feature.
Verify that the Gen5 Fluorescence/Luminescence filter table matches the
contents of the filter wheels. Verify that the EX filter in position #2 does not
overlap with the EM filter in position #3.
For models with injectors, the internal chamber may require cleaning (see
Preventive Maintenance).
Synergy 2 Operator’s Manual
184 | Appendix B: Error Codes
Code
Description and Possible Remedies
0700
0800
0900
Noise Test Errors, Offset Test Errors, Dark Range Errors
This series of System Test errors may indicate that there is too much light
inside the chamber. Make sure the reader’s shroud is properly fastened, and
the plate carrier door and the front hinged door are properly closed.
For models with injectors, if the dispense tubes are not connected to the
back of the reader, re-install the plastic plugs that shipped with the
instrument (or cover the holes with black tape). Restart the reader.
0E01–6
Fluorescence wavelength not found in table
This error indicates that the specified wavelength is not detected in the
reader’s filter table. The last number is the filter set number in the assay
protocol. In Gen5, verify the Fluorescence filter table has the wavelengths
loaded into the reader. Compare the contents of the table with the Excitation
and Emission filters installed (see the Gen5 Help system for more
information). Restart the reader.
0F01
Filter is defined in the wrong location
Ensure that Gen5’s Fluor/Lum wavelengths table matches the actual filters
installed in the filter wheels.
1306-7
1406-7
<Motor> not homed successfully
This error indicates that the <motor> is not at home.
Make sure the Plate Type described in the Gen5 Protocol matches the plate
you are using.
Check for any obstructions that may prevent the carrier, syringes, or filter
wheels from moving normally. Restart the reader.
2901
Tungsten Lamp reference voltage out of range
A test of the tungsten lamp is performed when the instrument is turned on
and then periodically during background functions. This error may indicate
that the lamp is weak or defective. Refer to the As Needed Maintenance
chapter for instructions to replace the lamp. If the error still appears after
replacing the lamp, contact BioTek TAC.
2A01
Plate jam error
Make sure the Plate Type described in the Gen5 Protocol matches the plate
you are using. This error can also occur if the plate type is correct but the lid
was left on the plate. If you wish to read the plate with a lid on it, create a
new plate type in Gen5 and be sure to enter the correct Plate Height.
Models with injectors: Verify that the tip prime trough and priming plate are
not stuck in the reading chamber.
2B01-04
Syringe motor axis did not find the home opto sensor transition
Generally, this error indicates the syringe was not properly installed. Make
sure the syringe’s thumbscrews are properly threaded. (Refer to the
Installation chapter for instructions.) Restart the reader.
2B0A
Priming plate not detected
Applies to models with injectors.
3306
Required carrier in when expected to be outside
The carrier is inside the read chamber and the probe needs to move down for
the requested operation. Press the Carrier Eject button and restart the
experiment.
BioTek Instruments, Inc.
Error Codes |
185
Code
Description and Possible Remedies
4xxx
PMT overload well error at <well #>
This error typically means that the fluid in a well has oversaturated the PMT
(i.e., the well is too “bright”). Try lowering the sensitivity value in the read
step.
Wells are counted starting at A1, moving left-to-right, row-by-row.
The row and column of the well can be extracted from the well number code
by applying the following formula (example uses 8 x 12 geometry, 96-well
plate):
1. Convert the ASCII hex string to a decimal equivalent.
Ex: “057” indicates 57 hex, yielding a well code of 87 decimal.
2. Row = (well code) / (columns in plate), rounded up to a whole number.
Ex: 87/12 = 7.25, indicating row 8 (or H).
3. Column = (well code) - ((row-1) * (columns in plate)).
Ex: 87 - ((8 - 1) * 12) = column 3.
NOTE: If this code is returned during an area scan, it indicates the scan point
corresponding to the row/column equivalent in the currently defined scan
map, NOT the actual well where the error occurred.
Synergy 2 Operator’s Manual
186 | Appendix B: Error Codes
BioTek Instruments, Inc.
Appendix C
Instrument Dimensions
for Robotic Interface
This section shows the location of the microplate carrier in
reference to the exterior surfaces of the Synergy 2 and the
mounting holes on the bottom. Use the illustrations to facilitate
system setup with a robotic instrument, such as the BioStack
Microplate Stacker. Dimensions are in inches.
™ The Synergy 2 is 17.5” D x 15.0” W x 17.5” H (44.5 cm D x
38.5 cm W x 44.5 cm H).
BOTTOM VIEW
The two arrows
point to special
mounting holes
for alignment
caps for
operation with
the Bio-Stack.
188 | Appendix C: Instrument Dimensions for Robotic Interface
Top View
Side View
If you purchased the BioStack to operate with the Synergy 2, the BioStack alignment kit
contains hardware for correct positioning. Refer to the Installation chapter in the BioStack
Operator’s Manual.
BioTek Instruments, Inc.
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