盤式分光光譜分析系統＿Gen5英文操作手冊

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Gen5™ Getting Started

Guide
Microplate Data Collection & Analysis

Software
BioTek® Instruments, Inc.

March 2009
© 2006–2009
PN 5321002
Revision G
BioTek® is a registered trademark and Gen5™ is a trademark of BioTek
Instruments, Inc.
Microsoft®, Internet Explorer®, Windows®, Windows XP, Windows 2000,
Windows Vista®, and Excel® 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.
Contents | i
Contents
Install Gen5 ..................................................................1

Gen5 License Agreement and Warranty Information.... 2
System Requirements............................................. 4
Installing the Software............................................ 6
Register with BioTek............................................... 9
Initial Setup ..................................................................13

Connecting a Reader .............................................. 14
Setting Up Gen5 and Gen5 ELISA ............................. 18
Setting Up Gen5 Secure .......................................... 22
Setting Up Gen5 Reader Control............................... 39
Getting Started..............................................................41
Getting Started...................................................... 42
Welcome Screen .................................................... 44
The Gen5 Workspace.............................................. 46
Introducing the Protocol Workspace.......................... 49
About the Menu Tree .............................................. 51
Buttons and Icons Guide ......................................... 52
Getting Technical Assistance.................................... 56
Essential Concepts ........................................................57

Experiment vs. Protocol .......................................... 58
About File Storage.................................................. 60
Best Practices........................................................ 62
Basic Tasks ...................................................................65

Quick Read ........................................................... 66
ii | Contents
How to Create a Standard Curve .............................. 67

Viewing Results ..................................................... 68
Printing Results ..................................................... 74
Testing the Reader ................................................. 75
Setting Up a Protocol ....................................................77

Design a Protocol ................................................... 78
Defining the Reading Procedure ............................... 79
Defining the Plate Layout ........................................ 81
Setting Up Data Reduction ...................................... 84
Customizing Data Views, Reports, and Exports ........... 86
Building Reports .................................................... 87
Exporting Results ................................................... 90
Sample Protocols ..........................................................91

How Do I Set Up My Assay?..................................... 93
Quantitative ELISA Example .................................... 94
Dual Wavelength Absorbance................................... 98
Basic Fluorescence Assay Example ........................... 101
Kinetic Fluorescence Assay Example ......................... 104
Fluorescence Polarization Example............................ 107
Max Binding Determination Competitive Assay ........... 109
Toxicity/Cytotoxicity Assay ...................................... 113
Dispensing Reagent................................................ 117
Troubleshooting ............................................................121
Troubleshooting ..................................................... 122
Communication Errors ............................................ 123
Restoring Optimal Performance ................................ 125
Fluorescence/Luminescence Measurements................ 126
Contents | iii
System Administrator’s Password ............................. 130

Database Errors ..................................................... 131
Index ............................................................................137
iv | Contents
Install Gen5
An “install wizard” guides you through the installation of Gen5; just

follow the prompts. But, before doing so, make sure your computer
and BioTek instruments meet the System Requirements outlined in
this section. You may also want to review the installation options,
software warranty, and related information.
Gen5 License Agreement and Warranty Information ........... 2

System Requirements .................................................... 4
Installing the Software................................................... 6
Register with BioTek ...................................................... 9
2 | Install Gen5
Gen5 License Agreement and Warranty

Information
License Agreement
BioTek Instruments, Inc. (also referred to as “BioTek”) has developed Gen5

Microplate Software for Windows. The price paid for this software includes the
license granting the purchaser a non-exclusive use of the Gen5 product per the
guidelines given below.
Gen5: The purchase of Gen5 entitles the installation of the licensed copy on up
to five (5) computers for simultaneous use. The installation of Gen5 on one
computer for the purpose of controlling one or two BioTek microplate readers
at a time is expressly permitted. Simultaneous use of the software on four other
computers solely for the purpose of analyzing, reporting and otherwise
working with data already collected is expressly permitted.
This license agreement does not grant the user title to the software or any
copyrights of proprietary rights in the software. The user may not sublicense,
rent, lease, modify, translate, decompile or disassemble the software for any
purpose. The license is not a sale of the original software or any copy.
Limited Warranty
BioTek warrants that for a period of ninety (90) days from the date of shipment,
the software product manufactured and sold by BioTek will conform, as to all
substantial operational features, to BioTek’s then current published
specifications (meaning those specifications in effect as of the date of purchase),
and will be free of defects which, in BioTek’s sole judgment, substantially affect
system performance. This warranty is limited to the original purchaser and
cannot be assigned or transferred. All claims under this Limited Warranty must
be made in writing to BioTek, Attention: Service Department or to BioTek’s
authorized representative or dealer. The purchaser must notify BioTek within
ninety (90) days after the date of shipment of the software to the purchaser of
any claim under this limited warranty. This 90-day period shall not be
extended by the delivery or receipt of any subsequent modifications to the
software. Modifications to the software within a version (“Updates”) are
Gen5 License Agreement and Warranty Information | 3
expressly excluded from the terms of this warranty. If the software is found to
be defective by BioTek, BioTek’s sole obligation under this warranty is to use
efforts consistent with BioTek’s regular business practices to attempt to remedy
such a defect. In no event shall BioTek’s liability under this Warranty exceed
the original purchase price of the software.
This warranty shall be null and void if the purchaser makes any modifications
to the software. Correction of difficulties of defects traceable to Purchaser’s
errors or system changes shall be billed to the purchaser at BioTek’s standard
time and material charges.
4 | Install Gen5
System Requirements
Before installing Gen5 make sure your hardware meets the minimum
requirements.
Computer Requirements
Minimum Requirements*: For Gen5 to run successfully, the computer must
meet the following requirements:
• Windows XP, Windows 2000, or Windows Vista (Professional Editions)
• Pentium III-Class PC (or compatible) processor (500 MHz or higher)
• 512 MB RAM or higher
• 2 GB Hard Drive space or higher
• Designed for XGA Resolution at 1024 x 768 or higher
• CD-ROM Readable Drive
• Keyboard
• Mouse
• Microsoft Internet Explorer v 5.0 or higher (for online Help)
• Microsoft Excel 2000 or later for Quick Export and Power Export
• Serial or USB port for BioTek instrument
* Note: Gen5 will run on a computer system that meets the minimum
requirements for most applications. For very fast kinetic reactions in multiple
wells, especially in higher density plates where large amounts of data are
captured, BioTek recommends a 2 GHz processor speed and at least 1 GB RAM
for best performance.
System Requirements | 5
Reader Requirements
Verify that the basecode built into your BioTek reader is compatible with Gen5.
BioTek has validated the following list of basecodes. Any instrument with this
basecode or higher is compatible with Gen5.
If your instrument reveals a basecode with a version number

lower than those provided here, please contact TAC for
instructions for downloading and installing updated software.
Instrument Basecode
PowerWave 1.21.1
PowerWave XS 1.06 (Any PowerWave XS2 basecode)
Synergy Mx Any Synergy Mx basecode
Synergy 4 1.03
Synergy 2 1.03
Synergy HT 2.24
FLx800 1.15
ELx800 3.07
ELx808 3.15
µQuant 2.02
Clarity NA, but version 4.0.1.10 or higher of Clarity

software is required
6 | Install Gen5
Installing the Software
Prerequisite
Gen5 requires the user who is installing the application to have Administrator
privileges for the Windows Vista, Windows 2000, or Windows XP system. If a
user with restricted access attempts to install the application, errors may occur.
Contact your organization’s system administrator if you are uncertain about
your privileges.
BioTek strongly recommends running a Windows Update to

ensure the latest Windows security fixes and critical updates are
installed prior to installing Gen5.
To install Gen5, follow these steps:

1 Start Windows.
• Be sure you have administrative privileges.
2 Place the Gen5 CD in the CD-ROM drive.
• If the CD does not launch the Install Wizard automatically,
complete Steps 3–5; otherwise, follow the Install Wizard
instructions.
3 Click the Windows Start button and select Settings>Control
Panel>Add/Remove Programs.
4 When the dialog box appears, click Add New Programs.
5 Click the CD or Floppy button and follow the prompts.
6 The Typical installation option is strongly recommended for most
users (see below).
7 Gen5 asks for the Serial Number shown on your product packaging.
Enter it and click Continue to save time later. If the number is
unavailable you can click Cancel and provide this information later.0
Installing the Software | 7
Be sure to register with BioTek for the fastest response from our support
team, should the need arise.
Custom vs. Typical Installation

The Typical installation option is recommended for most users. It installs:
• Gen5 application (Gen5.exe and supporting files)
• Manuals: PDF versions of the complete Gen5 User’s Guide
• Tutorials: animated demonstrations provided as a learning tool and
available from the Help menu
• Gen5 Diagnostic module
You can use the Custom installation option to prevent installation of the
Tutorials. If disk space on the computer is limited, for example, select the
Custom option during installation.
Click the down arrow next to a feature to display the options menu. Select the
desired option. When you opt to not install a feature, its disk icon is replaced
with a red X.
8 | Install Gen5
Gen5 OLE Automation Toolkit

Custom installation is required during software installation to install the Gen5
OLE Automation Toolkit for programming robotic instruments to use Gen5.
Instead of selecting the Install Wizard’s default option for Typical installation,
select Custom. Change the setting for OLE Automation Toolkit to install this
feature.
Gen5 installs an OLE Automation folder when this option is selected. You’ll
find the Gen5 Automation Programmer’s Guide (in PDF format), the
BTIStatusCodes.h file, and a Samples folder containing several program
samples in common programming languages.
To learn about security options when using OLE Automation,

see Login/Password Controls in the Gen5 Help.
Install Other Software Components

If you purchased the Gen5 QC Trending Module, you can install that software
now.
Next Steps: Register Gen5 and Initial Setup

After registering your software with BioTek, review the recommended initial
setup suggestions for the level of Gen5 you’re running.
Register with BioTek | 9
Register with BioTek
The Pre-Registration screen will appear when you launch Gen5 until the
software is registered. A trial-version serial number can be used for the
specified number of days until a licensed version is purchased. Unless the
purchased version of Gen5 is a higher version than the trial version there is no
need to reinstall the software; simply register the software.
As required for installing the software, you must have

administrative privileges to register with BioTek. Generally, the
user who logged in to Windows when installing Gen5 should be
logged in when registering the software.
10 | Install Gen5
Vista users: If prompted for administrative privileges, engage

them before registering the software: Locate and right-click the
Gen5 desktop icon, and select Run as administrator. At the
User Account Control dialog, click Allow.
Launch Gen5 and Register the Software

1 Open Gen5 by clicking its desktop icon or by using the Windows
Start button and selecting Programs > Gen5 > Gen5.
2 At the Pre-Registration dialog enter the product serial number (if it
wasn’t entered during installation or if you’ve been using a trial
version).
3 Click Register to register the software and receive a password. The
Registration dialog will appear with the serial number and the site
key; this is information provided by your computer.
Click Demo to run Gen5 without registering it for the number

of days displayed below the Demo button.
4 Click Go to Internet Site.

• Alternatively, if your Internet browser and Gen5 are on
different computers, enter the BioTek URL, shown above, into
your browser’s address field. Make note of the serial number
and site key to proceed.
If you do not have access to the Internet, contact BioTek (see

page 56).
Register with BioTek | 11
• When using the same computer, you can copy and paste
(Ctrl+C and Ctrl+V) the serial number and site key into the
registration form.
5 At BioTek’s Software Registration website page, enter or paste the
serial number and site key information into the form and click
Submit.
A registration form will be displayed containing any information
BioTek already has about you and your organization.
6 Review and edit the information as necessary, then click Submit
Registration Form.
7 Your password will be displayed on screen. Copy or make note of it.
8 Return to the Gen5 Registration dialog to paste or enter it into the
Password field.
9 Click Validate Password. 0
The software should now be registered and you will not see the
Pre-Registration screen again.
Gen5 stores the serial number and site key in the Help>About
Gen5 screen so you can log in to BioTek’s product registration
site at any time.
12 | Install Gen5
Initial Setup
The first thing all users must do after installing Gen5 is connect a
reader to the computer and tell Gen5 how to communicate with it.
Other initial setup steps can be performed to improve your
experience using Gen5.
Gen5 Secure, to meet FDA submission criteria, requires establishing
security conditions. You’ll find instructions for performing all these
tasks in this section.
Connecting a Reader ..................................................... 14

Setting Up Gen5 and Gen5 ELISA .................................... 18
Setting Up Gen5 Secure ................................................. 22
Setting Up Gen5 Reader Control ...................................... 39
14 | Initial Setup
Connecting a Reader
System > Reader Configuration

After following the operator’s manual instructions for attaching the reader to
the computer, you must tell Gen5 what type of reader it is and which
communications port (Com Port) it is plugged into.
Special note for Clarity users: Settings can be defined only

through the Clarity PC software. Refer to Clarity instructions on
page 16.
1 Turn on the reader.

2 From the Welcome screen (page 44), select System Menu.
3 From the menu bar, select System> Reader Configuration.
4 Click the Add button to define the Reader Settings.
5 Use the drop-down list to select the Reader Type.

6 Except for the Clarity, in the Com Port text field, enter the number of
the communications port. See note below about USB connections.
7 Retain the default Baud Rate.
8 Click Test Comm. Gen5 will attempt to communicate with the
reader.
9 After you receive a passing message, “The reader is communicating,”
click OK and then Close at Reader Configuration. If you receive any
other message, look for a remedy in the Troubleshooting section of
this guide.0
That’s it: Gen5 captures the information it needs from the reader itself,
including probe size, wavelength, and bandwidth capability, and any other
applicable information.
USB Connection: Plug in the USB cable and turn on the instrument:
• For first-time setup, Windows may recognize New Hardware and

prompt you to install the required driver. Follow the installation
Connecting a Reader | 15
directions provided with the driver. When the wizard is finished

installing the driver, a new “COM port” will be available. Note: This
step may not be needed.
• Go to Control Panel > Administrative Tools > Computer
Management > Device Manager > Ports (COM & LPT) to see
the new virtual Com port assigned to the USB. Enter this port for the
Reader Configuration.
Instrument-Specific Information
• Synergy HT: For this multi-detection reader, Gen5 must obtain the
fluorescence/luminescence filter sets. BioTek sets the reader’s on-
board software with the ordered configuration before shipping the
reader. Gen5 will capture the stored information when it initiates
communication with the reader.
• Synergy 2 and Synergy 4: For these multi-mode, multiple-detection
readers, Gen5 must obtain both the fluorescence filter sets and the
mirror-holder configuration. BioTek sets the readers’ on-board
software with the ordered configuration before shipping the reader.
Gen5 will capture the stored information when it initiates
communication with the readers.
• Synergy Mx: For this multi-mode reader, BioTek sets the reader’s
onboard software with the ordered configuration before shipping.
Gen5 will capture the stored information when it initiates
communication with the reader.
• Clarity: A Clarity luminometer can be added or deleted in the
instrument configuration dialog. But, unlike other readers, the COM
port that is used to attach to the Clarity cannot be set or modified in
the Gen5 Reader Configuration dialog. You must use the Clarity
software to define the connection settings. Use the Clarity Control
Panel.
• FLx800: Gen5 must obtain the fluorescence/luminescence filter
cartridge configuration. BioTek sets the reader’s onboard software
with the ordered configuration before shipping the reader. Gen5 will
capture the stored information when it initiates communication with
the reader.
16 | Initial Setup
• PowerWave: This eight-channel monochromator does not need

special setup. When Gen5 communicates with the reader the
Absorbance Wavelength table currently stored in the reader’s memory
is displayed. When defining a Read Step, select from the stored
wavelengths or enter a different one.
• PowerWave XS: This single-channel monochromator does not need
special setup. When Gen5 communicates with the reader the
Absorbance Wavelength table currently stored in the reader’s memory
is displayed. When defining a Read Step select from the stored
Two models of PowerWave XS are listed in Gen5: PowerWave

XS and PowerWave XS2. If you are connecting a PowerWave XS
reader that has a USB port and an MQX200R2 product number
(take note of the 2), you must select the PowerWave XS2. Our
changes to the PowerWave XS hardware to incorporate a
USB/RS-232 COM port require unique reader identification in
Gen5. There is no difference in the optical performance
characteristics of the reader.
• ELx800 and ELx808: BioTek configures the onboard software of these

filter-wheel-based readers with the installed filters. Gen5 will capture
the filter configuration when it initiates communication with the
reader.
• µQuant: This single-channel monochromator does not need special
setup. When Gen5 communicates with the reader the Absorbance
Wavelength table currently stored in the reader’s memory is
displayed. When defining a Read Step, select from the stored
Setting Up the Clarity Luminometer
Clarity users must follow a slightly different sequence of steps to establish

communication between the Clarity and Gen5. First, follow the installation
instructions provided with the Clarity, including installing the Clarity PC
software. After installation, when you’re running assays, Gen5 uses the Clarity
Connecting a Reader | 17
PC software in place of its StepWise Procedure. But, you define the other
elements of the Protocol and run the Experiment with Gen5.
Install the Clarity PC software before proceeding.
1. Connect the luminometer to the computer (if not already connected)

and turn it on.
2. Start the Clarity software and set up the communications port
(Options > Com Port Settings).
3. Then, select Options > Instrument Info to test communication.
Details about the current instrument should be displayed on screen. If
not, repeat Step 2, making sure the correct port is selected.
4. Important: Close the Clarity software.
5. Start Gen5 and log in (if required).

6. Select System> Reader Configuration and set the Reader Type to
Clarity.
7. Select System> Reader Control> Clarity to make sure Gen5 is
communicating with the Clarity. If the control panel does not open,
repeat Step 6 and retry. Contact BioTek TAC if problems persist.0
If the control panel does not open:
• on Windows XP and 2000 systems, repeat Step 6 and retry.
• on Windows Vista systems:
1 Locate and right-click the Clarity desktop icon, and select
Run as administrator. If prompted, enter the password.
2 At the User Account Control dialog, click Allow. The
Clarity software will launch and communicate with the
reader.
3 Close the Clarity software.
4 Return to Gen5 and test communication with the Clarity
by performing Step 7 above.
• Contact BioTek TAC if problems persist.
18 | Initial Setup
Setting Up Gen5 and Gen5 ELISA
For Gen5 and Gen5 ELISA Only
Gen5 fulfills the reader control and analytical needs for a wide range of
laboratory settings. The degree to which you follow the recommendations
provided here depends on the needs of your organization.
Recommended Tasks to Perform

1 Designate a System Administrator.
2 Install Gen5 on the Administrator’s computer.
3 Change the System Administrator’s password.
4 Determine the optimal way to store Gen5’s protocol and experiment
files: File Storage (page 20).
• Organize the database or your Windows file structure.
5 Install Gen5 for other users and connect a reader to each computer
(page 14).
6 If applicable, direct each user’s database configuration to point to the
correct shared database.
7 Learn how to use Gen5: 0
• Check out the tutorials in the Help system: Select Help >
Tutorials.
Setting Up Gen5 and Gen5 ELISA | 19
Gen5 System Administrator
For all levels of Gen5 except Gen5 Secure and Gen5CL

It is necessary to log in as the System Administrator to change user permissions
and database configuration, and to access any features that are denied as Users
Permissions.
How to Change the System Administrator’s Password
This function is available only to the System Administrator. You

must log in as the Administrator (System> Administrator
LogIn) to access these controls.
1 Select System> User Setup, and select the Administrator tab.

2 Select a Startup Action and Protocol Folder, if desired.
3 Enter the current password in the Current Password field. Gen5 ships
with the password set to admin.
4 Enter the new password in both the New and Confirm password
fields.
5 Click OK.0
How to Maintain Users’ Permissions

Except in Gen5 Secure, access to Gen5 functions, like reading a plate, modifying
a protocol, and masking values, is defined equally for all users except the
System Administrator, who has all permissions.
20 | Initial Setup
File Storage
System > Preferences > File Storage Mode

Use this control to select a method for storing protocol and experiment files.
Attention Gen5 Secure users: To ensure 21 CFR Part 11

compliance use the Gen5 Database for file storage.
About File Storage

Gen5 provides two methods for storing Protocol and Experiment files. You can
use the secure, shared-access database provided with Gen5, which is required
for compliance with the FDA’s 21 CFR Part 11 regulation on electronic records
submission. Alternatively, you can use the file system provided with the
Windows operating system on a local PC or network (LAN). If your
organization is unconcerned with FDA regulations, the choice is a matter of
preference. However, one advantage to using the Gen5 Database is its ability to
recover from a system crash. New and modified files are saved as temporary
files in the database and can be used to recover information that wasn’t saved
before a system failure.
How to
Select an option for storing Experiment and Protocol files:
• Gen5 Database: all actions related to managing and maintaining files,
like File > Open, File > Save, Browse, and so on, will occur in the Gen5
SharedDB.
• Windows File System: Gen5 will not control the management of files.
Actions related to managing and manipulating files will be determined by
the Windows operating system; for example, you can use Windows
Explorer. Generally during file management activities like File > Open,
Windows begins at the last directory and folder used.
AutoSave Feature: Gen5 offers this feature to give you

additional control over the storage of saved files.
Setting Up Gen5 and Gen5 ELISA | 21
Gen5 User Permissions
For all levels of Gen5 except Gen5 Secure and Gen5CL

Only Gen5 Secure product level offers the multiple-user login and password
controls required to comply with the FDA’s electronic-records submission
directive. This page describes the more limited security options provided in
other Gen5 products.
About Gen5 User Accounts

There are only two types of users in Gen5: Administrator and User, that is,
System Administrator and non-administrator. Neither account can be deleted.
You Can: You Cannot:
Change the Log in as a User; only the System

Administrator’s password Administrator requires login
Change the permissions Change the permissions for

for User Administrator; all rights and
privileges are already given
Maintain/store Gen5 files Keep track of which user is

in a secure database responsible for Gen5 activities
Select a Startup option and Turn on/off Audit Trail notification;

Protocol and Experiment events for which users are invited to
Folders for users add comments to the change
log/audit trail are fixed by Gen5
22 | Initial Setup
Setting Up Gen5 Secure
Follow these procedures to set up Gen5 Secure:

1 Designate a System Administrator.
2 Complete the System Administrator’s To-Do List (the Initial Setup
tasks) (next page).
3 Organize the database (page 26).
4 Review/modify Signature Reasons and other security controls (Select
System > Security).
5 Define certain preferences by using the Gen5 Default Protocol. (Each
shared database has one Default Protocol which defines the initial
settings for all newly created protocols.)
6 Install Gen5 (and the QC Trending module, if applicable) on each
user’s computer.
7 Set up each user’s database configuration to point to the correct
shared database (page 28).
8 Connect a reader to each user’s computer.
9 Advise users to change their passwords and encourage them to watch
the Gen5 Tutorials.0
Setting Up Gen5 Secure | 23
System Administrator’s To-Do List
For Gen5 Secure and Gen5CL only
Initial Setup Tasks

1 Make sure all designated computers (PCs) and BioTek readers meet
the Minimum Requirements (page 4).
2 Install Gen5 Secure on one computer (PC).
3 Start Gen5 and log in as the System Administrator.
4 Change the System Administrator’s password.
5 Copy the database Shared.mdb to a secure network location.
6 Test Database Configuration of the Shared.mdb on the network.
7 Create/modify User Groups, as needed, and assign User Permissions
to the Groups.
8 Create new User Accounts and assign the users to a Group.
9 Connect reader(s) to the PC and establish communication.
10 Repeat Steps 2, 3, 6, and 8 for the remaining PCs.0
Install the Gen5 QC Trending Module on all the PCs as well, if
applicable.
Periodic/As Needed Tasks

• Customize the security features to accommodate your organization’s
needs.
• Organize your database files.
• Educate users on regulatory requirements and Gen5 best practices.
• Establish and implement a procedure and schedule for record retention
and archival.
• Review records, including any training/user-qualification records.
Before modifying a user’s account, make sure he/she is not

logged in to the system. You can check the System Audit Trail
to determine who is currently logged in.
24 | Initial Setup
Change the System Administrator’s Password
System > Security > Users

BioTek recommends changing the System Administrator’s password
immediately following Gen5 installation to ensure a secure operating
environment.
To change the password:

1 Log in as the System Administrator, if you haven’t already done so.
• Select System > Login/Logout.
• Set the User to Administrator.
• Enter the default password: admin.
2 Select System > Security > Users.
3 Double-click the System Administrator user (to edit the record).
4 Define and confirm the new password. The System Audit Trail will
open to log the change and accept your comments. 0
Do not forget the Administrator’s password. If you do, you’ll

have to reinstall Gen5.
About Gen5 Databases
All levels of Gen5 install two databases during regular installation: SharedDB
and LocalDB. (Learn more about Gen5 databases in the last section of the
guide.)
• SharedDB can be set up on a network for sharing information among
multiple users. It contains all protocol and experiment data files and their
associated audit trails, the plate types, and reader-diagnostic history data.
In Gen5 Secure, SharedDB also contains security settings, user accounts,
and a system audit trail for shared events. This database can be moved,
renamed, and copied. So, if desired, you can create a unique database for
individual projects, teams, or other classification.
The QC Trending database, installed with the QC Trending

module, resembles the SharedDB. It can be set up on a
network for sharing among multiple users, moved, renamed,
and copied. It is initially installed in a QC folder in the default
database location described below.
• LocalDB contains the local setup information, including the Reader

Configuration. For Gen5 Secure, this database also contains an audit trail
for local events. LocalDB is stored on the computer’s hard drive, and it
cannot be moved or renamed.
• Default database location: During normal installation Gen5 installs its

databases in the Windows Common Application Data Folder:
• Windows XP and 2000: C:\Documents and Settings\All
Users\Application Data\BioTek Instruments\Gen5 (software
edition)\(version #)\SharedDB or LocalDB
• Windows Vista: C:\Program Data\BioTek \Gen5 (software
You may need to change your operating system settings to view

the Common Application Data folder.
• Max Size: the maximum size of the database files is 2 gigabytes (GB). At
startup, Gen5 checks the remaining size of the database. Warning
messages are displayed when the database size exceeds 1536MB. Use the
Gen5 backup and maintenance features to archive your database records.
• Gen5 has built-in error recovery modes. When your connection to the
database is lost for any reason, Gen5 saves any unsaved files as
Temporary Files. After a system failure, the next time you open an
affected protocol or experiment file, Gen5 offers to replace the unsaved
files with the Temporary Files. Select Yes to recover any changes made to
the files before the system failure; select No to open the files as they were
last saved, before the unsaved changes were made. Newly created files
are saved as Temporary Files, also. Following a system failure, you can
rename these temporary files with the proper filename extension (.xpt or
.prt) using Gen5 Maintain Files controls.
26 | Initial Setup
• Gen5 offers automatic backup: you can define settings for regularly
and automatically backing up and optimizing databases with the Gen5
periodic Optimize feature.
Learn more about the databases in Gen5 Help.
Organize Your Database Files
During regular installation, Gen5 Secure installs and enables the

shared database to store experiment and protocol files.
All of your file management requirements can be fulfilled using Gen5

databases. You’ll be most satisfied with the final structure if you spend some
time planning it up front. In a multiple-user environment, you can set up a
Gen5 database on a shared-network drive (LAN) so multiple users can access
the same protocol and experiment files, including the Default Protocol.
Multiple Databases: You can create multiple copies of the clean, installed
SharedDB, renaming them with meaningful titles for use by various projects or
teams or researchers. Within each database you can set up a consistent file
structure, for example, specific folders for specific types of Protocols and
Experiments, or a different folder for each user. The possibilities are endless.
Backups: Performing backups on a regular schedule is highly recommended to
preserve your data. And, Gen5 provides a tool to schedule backups to occur
periodically. See below.
L File Management Recommendations

• Put a copy of the SharedDB on a shared-network drive where all your
Gen5 users can access it. Be sure to set each user’s Database Configuration
to point to the correct location.
• Consider setting up shared databases for different projects or teams
within your organization. You can follow the steps defined earlier to
create multiple databases in the same folder (or directory), or you can
move the unique databases to a different network location/folder. Use
Database Configuration to point users’ Gen5 sessions to the correct

database.
• Regularly archive or back up the database to preserve your records. There
are numerous ways to do this; BioTek recommends following your
organization’s existing policy for securing data. For example, if you put
the shared database on the network and your network is backed up every
night, this may be sufficient. You can use Gen5 Optimize and Backup
Settings to facilitate your data-protection policy.
• Gen5 handles multiple, simultaneous users performing database
management tasks by giving precedence to the user with the greater
administrative rights.
Copy the Shared Database to a Network
Use Windows Explorer to move a copy of the SharedDB to a shared network

location.
• Before moving the SharedDB to a network location, make a copy of it to
use as a template for future use.
1 In the default SharedDB folder, highlight the original database, right-
click it, and select Copy.
2 Deselect the original file (click elsewhere in the dialog), right-click,
and select Paste.
3 Highlight the copy, right-click, and select Rename.
4 Give the copy a unique name, like SharedDB_original.mdb.
• QC Trending Module: If you have installed the QC module, BioTek

recommends handling its database similarly. Make and save a clean copy
of the QCDB.mdb, and move one copy to a shared network location.
28 | Initial Setup
Configure PCs to Use the Shared Database

You must configure each user’s PC (computer) to point to the correct database.
1 Log in as the System Administrator (System > Login/Logout).
2 Select System > Database Configuration and select the Shared
tab.
3 Click the 3-dot button next to the Source field and browse to the
secure network location of the SharedDB.
4 Click Test to make sure the connection is accurate. 0
Repeat these steps for the QC Trending database, if applicable.
About User Accounts
Prerequisite
This function is available only to the System Administrator. You must log in as
the Administrator (System > Login/Logout) to access all the controls. Non-
administrators are limited to changing their own password and selecting a
Startup Action and Protocol Folder.
How to Create, Modify, or Delete User Accounts

Only an Administrator can add, modify, or delete users. You can designate
multiple individuals to be system administrators. Any user account can be
changed or deleted, except there must always be at least one System
Administrator:
• Click New to set up a new user.
• Double-click or highlight a user and click Edit to modify its

name, password, or Group assignment.
• Highlight a user and click Delete to remove the user account.
About User Groups
System > Security > Groups
Prerequisite
the Administrator (System > Login/Logout) to access this control.
Gen5 Secure uses Groups to manage the rights or permissions granted to users.
When creating (or maintaining) a group, you define the level of access and the
controls available to certain types of users, and then assign actual users to the
groups. Gen5 ships with three groups: Administrator, Power User, and
Standard User.
The System Administrator and Power User groups are given access rights to all
functions. The Administrator’s rights cannot be changed, and include
additional rights to manage user accounts that are not extended to Power
Users. When Gen5 Secure is installed, the Standard User is limited to the
following permissions. The System Administrator can change these rights as
needed:
• Open Protocol
• Add a New Plate
• Create/Edit Sample IDs
• Edit Plate Information
• Edit Plate Layout
• Edit Report Builder
• Create folder in database
• Define test plate
30 | Initial Setup
How to create new and modify existing groups:

Only a System Administrator can add, modify, or delete groups. Except for the
Administrator group, any group can be changed or deleted, and any group can
be renamed.
• Click New to set up a new group.
• Highlight a group and click Edit to modify its name and

permissions.
• Highlight a group and click Delete to remove it as an option.

First you must reassign any users to another group. You cannot delete a
group with users assigned to it.
Creating/Maintaining User Accounts
Prerequisite
Most options for user accounts are available only to the System Administrator.
Non-administrators are limited to changing their own password and selecting a
Startup Action and Protocol Folder.
User ID
Enter a unique ID using 1 to 16 alphanumeric characters. The user will enter or
select this ID when logging into Gen5 and when signing files.
Full Name
Enter the user’s name. This name will be associated with events logged by this
user’s actions and with the digital signature applied by this user.
Group
Choose a Group membership to assign access rights and permissions to the
user. Users receive the rights assigned to the Group.
Status
The checkbox shows whether the user’s account is currently locked. The System
Administrator can lock or unlock the account. When a user’s account is locked,
the user cannot log into Gen5 and cannot sign files. A user’s account may
become locked due to one of three events:
• Intentional lock by the Administrator through this dialog
• Automatic lock if the user exceeded the number of successive failed login
attempts
• Automatic lock if the user’s password expired
Unlocking a user’s account following an automatic lock resets its

counter or clock. The reset is specific to the reason for the
lockout: when it is caused by password expiration, the password
expiration clock is reset; when it is caused by failed logins, the
user’s history of “successive failed login attempts” is reset to 0.
Startup Action
Use the drop-down to select the preferred method for starting Gen5:
• Startup Window is the default setting. It offers several options including
creating a new item or opening a recently used item.
• Create new experiment opens Gen5 with the Protocol selection dialog
open, as if the user had selected File > New Experiment.
• Start at main menu opens Gen5 showing the File, System, and Help
menus only. Since neither a protocol nor experiment is open, the
workspace is blank.
Protocol and Experiment Folders

Browse to or enter the full path and directory to define the folder in which the
current user will typically store protocol and experiment files. If a folder is not
specified, Gen5 will default to the most recently accessed folder.
Password
Assign a password for the user to enter the first time he/she logs in to Gen5.
Instruct users to change their password after the first login using the password
32 | Initial Setup
you’ve assigned. Users can change only their own password. System
Administrators can change any user’s password.
Login/Password Controls
System > Security> Login

Prerequisite
Only the System Administrator can access these controls. You must log in as
the Administrator (System > Login/Logout) to change the settings.
The default settings shipped with Gen5 Secure, and shown in

the screenshot below, comply with the FDA’s 21 CFR Part 11
requirements for controls for identification codes/passwords.
Login
• Lock user account after: Specify the number of successive failed
login attempts a user may make before being locked out of Gen5. This
feature does not apply to System Administrator accounts, and only a
System Administrator can reinstate a locked-out account. Valid entry
range: 2–10. When this feature is not selected, users’ login attempts
are unlimited. Compliance with 21 CFR Part 11 requires setting a limit
for failed login attempts.
• Lock session after: Specify the number of minutes that a Gen5
session can be idle before it is locked and requires successful user
login to reactivate. A session is considered idle when there is no
keyboard or mouse activity and Gen5 is not controlling a reader
activity. Valid entry range: 1–1440 minutes. Compliance with 21 CFR
Part 11 requires setting an idle-time limit.
• Force user to type ID: apply this control if your security rules
require users to enter their ID at login and to apply their Signature.
When this feature is not selected, the last user’s ID is displayed in the
login and signature screens and users can select an ID from a drop-
down list of users. This is not a requirement for compliance with 21
CFR Part 11.
• Require login in OLE Automation (BioStack): select this option to
ensure that Gen5 security permissions are enabled when Gen5 Secure
is run as an OLE Automation server, for example, for using the
BioStack. If Windows Authentication is enabled, the login process
may take place automatically. If the user does not have Windows
Authentication enabled, Gen5 prompts the user to enter a login ID
and password. Logins performed while this option is selected are
tracked in the Audit Log.
Password
• Minimum password length: Specify the minimum number of
alphanumeric characters required for a valid password. Valid entry
range: 2–10 characters.
• Password expiration: Specify the number of days a password can be
used before users are required to change it. When users let their
password expire without changing it, their accounts are locked out
34 | Initial Setup
and only a System Administrator can reinstate a locked-out account.

Valid entry range: 1–10000 days. If this feature is not selected,
passwords do not expire. Compliance with 21 CFR Part 11 requires an
expiration period.
• Lock out: when a user’s password has expired, the system
administrator has two choices:
• Manually remove the Locked out flag. This resets the
password expiration period allowing the user to log in
using his/her current password.
• Enter a new password for the user (which unlocks the
account) and tell the user to log in with the password you
have assigned him/her. Advise the user to change the
password after logging in.
• Advise user: If password expiration is set, specify the number of days
before passwords expire to alert users to change their password. Valid
entry range: 1–30 days, but cannot exceed the number of days to
Password Expiration.
• Password reuse: Specify the number of passwords Gen5 will
remember for each user’s account to prevent a recently used password
from being reused. Valid entry range: 2–20.
Configuring Windows Authentication
Gen5 Secure provides an option to allow users to sign in to Gen5 using

Microsoft Windows Authentication instead of user accounts defined in Gen5.
Through Windows Authentication, also known as single sign on (SSO), users
log in to their workstations once and have access to Gen5 without having to log
in again. The Windows Authentication feature benefits system administrators
as well by providing a single location for the management of user settings.
To use the Windows Authentication feature, workstations must be running
Windows XP or Vista; earlier versions of the operating system will not work
properly. Servers must be running Windows 2003 or 2008.
BioTek has validated this feature on Microsoft Windows Server

2003 (SP2 and SP3) and Microsoft Windows Server 2008 (SP1).
BioTek does not provide support for the use of the Windows
Authentication feature on a server that has not been validated.
Before Windows Authentication can be activated in Gen5, both the Gen5

Administrator and the IT administrator must perform some setup tasks.
Gen5 Administrator Tasks

Gen5 Secure uses Groups to manage the rights or permissions granted to users.
The Group Names are defined in Gen5 by the Gen5 Administrator.
When creating or maintaining a group, the Gen5 Administrator defines the
level of access and the controls available to certain types of users, and then
assigns actual users to the groups. Gen5 ships with three default groups:
Administrator, Power User, and Standard User.
The System Administrator group is given access rights to all functions. The
Administrator’s rights cannot be changed. When Gen5 Secure is installed, the
Standard User is limited to the following permissions (the System
Administrator can change these controls as needed):
• Open Protocol
• Use Gen5 Protocols (requires Gen5 Native)
• Add a New Plate
• Create/Edit Sample IDs
• Edit Plate Information
• Edit Report Builder
• Create folder in database
• Edit Paneled protocols
• View protected/read-only protocol items
36 | Initial Setup
Gen5 users assigned to Gen5 Groups
IT Administrator Tasks
The IT administrator must create user groups on the server to mirror the Gen5
User Groups defined in Gen5. The user groups on the server must be named
Gen5_<Gen5 Group Name>, where <Gen5 Group Name> is the name of a
group in Gen5, such as Gen5_System Administrators. The IT administrator can
also define permissions that apply to Windows resources, such as access to
folders, printers, and so on.
Gen5 Groups defined on the server
Each user who has permission to run Gen5 must be defined as a member of a
single Gen5 group in Windows. If a user is defined as a member of more than
one Gen5 group, an error message will appear when the user attempts to log in
to Gen5. If the user is not assigned to a Gen5 group, a warning message will
appear, indicating that the user is not a valid Gen5 user. The user can then log
in with another, valid Gen5 account.
Configuring the Email Notification Feature
Gen5 Secure can be set up to send an email notification to specified recipients

when a predefined event, such as a reader error or failed login, occurs. The
email server must be configured to support this feature. The Gen5
Administrator must test these settings to verify that the Email Notification
feature is functioning correctly.
To verify that the email server is configured correctly to support

this feature, check with your IT administrator.
In the Send Emails from area, you can define the email addresses used in the
Email Notification feature.
38 | Initial Setup
• Display name: This field is required. By default, Gen5 displays

Gen5_Notice_Do_Not_Reply@biotek.com as the address from which the
notification message is sent. This email address can be modified to any
valid email address.
• Forward errors to: The address specified in this field will receive any
mail delivery error messages generated by the server related to the Email
Notification feature. If no address is entered, error messages will be sent
to the address in the Display Name field.
• Reply to: The address specified in this field will receive messages sent as
a reply to an Email Notification message. If no email address is entered in
this field, the address in the Display Name field will receive any reply
emails.
Defining a Custom Email Template

By default, Gen5 generates the title and body of the email notification
messages, but you can define a custom template that includes specific variables.
1 On the Email tab of the Security dialog, click the three-dot button
in the Template column.
2 In the Email Definition dialog, select Custom as the mode and either
Text or HTML as the format.
3 Modify the Subject and Body text as necessary. Variables associated
with the defined event are listed in the Variables textbox. To insert
them in the Subject or Body of the email notification, place the cursor
where you want the variable to occur, then double-click the variable
in the Variable textbox.
4 Click OK.
Setting Up Gen5 Reader Control | 39
Setting Up Gen5 Reader Control
For Gen5 Reader Control Only
Recommended tasks to perform:

1 Install Gen5 on the computer.
2 Determine the optimal way to store Gen5 Protocol and Experiment
files: see File Storage (page 20).
3 Connect a reader to the computer (page 14).
4 Set user preferences.
5 Learn how to use Gen5: 0
• Watch the animated demos, select Help > Tutorials.
40 | Initial Setup
Getting Started
This section provides introductory information to help you learn how

to use Gen5.
Getting Started ............................................................. 42

Welcome Screen ........................................................... 44
The Gen5 Workspace ..................................................... 46
Introducing the Protocol Workspace ................................. 49
About the Menu Tree ..................................................... 51
Buttons and Icons Guide ................................................ 52
Getting Technical Assistance ........................................... 56
42 | Getting Started
Getting Started
Welcome to Gen5! To make you feel comfortable with this very capable reader
control and data analysis software, we’ve provided several learning tools.
Tackle them progressively and you’ll be a pro in no time. You’ll find some of
these tools in the Gen5 Help system.
These topics do not describe the initial setup requirements, like

connecting a reader. Unless your System Administrator has
already done so, complete the initial setup requirements before
proceeding.
Watch a Demo: Select Tutorials from the Welcome Screen

• Basic #1: Intro to Gen5
• Basic #2: Setting up the Procedure
• Basic #3: Defining the Plate Layout (not for Gen5 Reader Control)
• Basic #4: Creating Data Reduction Steps (not for Gen5 Reader Control)
• Basic #6: Building Reports
• Data Views: Create a New View
• Intro to Power Export (to use Microsoft Excel for reporting)
• How to create multiple standard curves
• How to determine optimal PMT Sensitivity
• Synergy 2 and Synergy 4: Filter-based FL Detection Review
Getting Started | 43
Open a Sample Protocol:

Gen5 ships with numerous protocols so you can load one up, customize it to fit
your particular requirements, and run an experiment.
1 Select File > Open Protocol.
2 At the top of the Open dialog, click the Up One Level button .
3 In the Gen5 folder or the DB (shared database), select the Samples
folder.
4 Select the folder for the desired detection method: Absorbance,
Fluorescence, or Luminescence.
5 Open the Protocol folder and select one. 0
Recommendation: Before making any modifications to the

sample protocols, BioTek recommends selecting File > Save
As after opening them and assigning a unique name to the
protocol. This will preserve the original sample protocol for
future use. Also recommended when saving a copy of the
original protocol is selecting the most convenient location for it.
You may want to use the Up One Level button a few times to
get back to the Gen5/Protocol folder, a more likely location for
your “in use” protocols. Note: The step-by-step instructions for
opening a sample protocol are based on the default filenames
and path.
The sample protocols must be considered as examples provided

for demonstration and guidance purposes. If you plan to use
these protocols or similar ones in a real application, it is your
responsibility to validate the protocol parameters, including the
report and export content (if applicable), before using them.
Your reader may not support all of the sample protocols

provided. Review the descriptions in the Sample Protocol and
Experiments Guide to see if your reader is compatible with the
defined steps. If you can read PDF files, click the Windows
Start button, select All Programs > Gen5 > Sample
Protocols and Experiments Guide.
Welcome Screen
Whenever you start Gen5 (unless your startup preferences are changed) Gen5
presents the Welcome screen. You can also return to the Welcome screen by
clicking File > Welcome Screen.
Instant Access
The Welcome screen provides quick links to give you instant access to:
• Sample protocols and experiments provided by BioTek
• Recently opened files: the four most recently used protocols and
experiments; click More to browse for an already-created file
• The Gen5 protocol Wizard, which helps you create a protocol and run
an experiment
Welcome Screen | 45
• The Read a Plate link, which performs a Quick Read

• The System Test, which can be run on the attached reader
• Self-running Tutorials
• The Gen5 menu: select System Menu to configure a reader, for
example
• A link to exit Gen5 (if the Welcome screen was opened by loading
Gen5)
• A link to close the Welcome screen (if opened by clicking File >
Welcome Screen from within Gen5)
The Gen5 Workspace
Gen5 offers several controls and workspaces for developing protocols, running
experiments, and viewing and reporting results:
1 Protocol
Every experiment is based on a protocol. The differences between a Protocol
and an Experiment in Gen5 are described in the Essential Concepts chapter.
2 Plate View
Gen5 provides a view or workspace for each plate processed (or to be
processed) in an Experiment. You must have an Experiment, rather than a
Protocol, open to have a Plate View.
The Gen5 Workspace | 47
To open the Plate View: In an Experiment, if it is not already open in the main
view of Gen5, double-click the plate icon in the menu tree or select
Plate > View.
Gen5 offers several ways to modify and customize the Plate View for on-screen
display and reporting/outputting results, see Viewing Results on page 68 to
learn more.
3 Menu Tree
The menu tree, docked at the left side of the workspace, provides a quick and
easy way to open all the tools needed to create and run experiments.
4 Toolbars and Menus

See Buttons and Icons Guide on page 52 to learn about Gen5 toolbars, buttons,
and menus.
5 Running an Experiment
All of the views and components come into play when running an experiment
(File > New Experiment). The Protocol menu tree is the primary engine for
an experiment, and Plates (one for each plate processed or to be processed) are
added to the menu tree. The Plate View and other plate components are made
available:
Information contains the text input at the Runtime Prompts when the
plate is read and BioCell™ results when the pathlength-correction option is
used.
Sample IDs are user-defined sample names or bar codes.
Calculation Log keeps track of and displays any data reduction errors.
Audit Trail logs changes (masking and editing) of data points for all levels
of Gen5, and numerous other events for Gen5 Secure. Audit trail events can be
included in reports and export files.
Introducing the Protocol Workspace | 49
Introducing the Protocol Workspace
When you create a new protocol, Gen5 opens a special workspace limited to the
protocol’s components:
The workspace is made up of the menu tree with a branch for each of the
protocol’s elements. The order of the protocol elements reflects the order to
follow when defining most protocols:
Defining the Procedure or reading parameters, like detection method,

wavelength, and other factors, is the most important step to Gen5. The
Procedure describes the data sets which are used in most subsequent steps to
generate results output. The Plate Layout is the only other protocol element
that is not affected by the Procedure.
For most protocols, it’s best to define the Plate Layout in your second step.
Gen5 automatically performs a blank-subtraction calculation when Blanks are
defined in the plate layout. (You’ll see this Transformation in the Data
Reduction workspace.) Defining the standards and their concentrations in the
plate layout is a prerequisite to generating a standard curve.
Data Reduction is one of the most powerful features in Gen5, and it

requires the information provided by the two previous steps to logically offer
you its capabilities. Automatically generated transformations, like pathlength
correction and the ability to conduct well analysis, for example, depend on the
Procedure. To plot a standard or titer curve and to validate Transformation
formulas requires knowing the Plate Layout.
Runtime Prompts are user-defined text fields that are presented to users
when they read a plate. The information obtained is stored in the Plate
Information component of the experiment and can be included in reports and
export files.
Viewing and Reporting Results: the next five options

(or three options if you’re running Gen5 ELISA or Reader Control, which do
not offer Power Export or Patient Report Builder) are tools for selecting and
customizing the appearance of data sets: raw data measurements and data
reduction results. Data Views controls the on-screen appearance of data. Data
Views also stores and makes available for reporting and exporting any
customizations made to the data sets. For example, you can build your own
matrix, table, or curve view of a data set by combining and formatting data
elements. When you do this in Data Views, that user-defined data set is
available for printing or exporting using the Report Builder, Patient Report
Builder, File Export Builder, and Power Export Builder.
Protocol Options provide several special features and preferences. Many

of the options may be rarely used by your organization. Review the options
provided and if your preferences vary from the default settings, use the default
protocol to set them for all newly created protocols. The exception to this rule
occurs with multi-plate protocols: calibrator-plate protocols and, for Gen5 and
Gen5 Secure users, multi-plate assay protocols. These types of protocols begin
by selecting the Protocol Type defined in the Protocol Options.
About the Menu Tree | 51
About the Menu Tree
• In an experiment, the menu tree is docked at the left side of the

workspace, unless its position has been previously altered. When you’re
working with a protocol file, the menu tree, like the toolbar, is limited to
related operations.
• The menu tree provides a visual cue of the steps to follow when creating a
protocol.
• All of the controls available from the menu tree can alternatively be
accessed using toolbar buttons or the drop-down menus.
• and symbols next to an item expand or close it to reveal or hide its

components.
• Highlight an item in the menu tree and right-click for a context-sensitive
menu of options, including Read when a plate is selected, for example.
• Asterisks (*) are displayed next to plate icons (and in the title bar) of an
experiment when a change is made or an action is taken but the file has
not yet been saved.
• You can move the menu tree to another corner of the workspace or let it
float undocked like the Plate workspace: click the undock button , drag
the title bar, and drop it in the desired location.
• When you Add multiple plates to an experiment, highlight a plate and
right-click for menu options to delete and renumber plates.
Buttons and Icons Guide
Button Description
The 3-dot (three-dot) button is a tool for customizing or

modifying the item’s contents; in some report options,
you must click in a field to activate its 3-dot button
Open an existing Experiment
Create a new Experiment or Protocol
Add another plate to the current Experiment
Read the plate (or Simulate, Manually Enter, or Import

data)
Save the Experiment
Open the Gen5 Wizard to help you create an Experiment
Print the results, but first use Report Builder to select

what to include
Print Preview
Reader Control: check the status, open the control panel
Duplicate View: click the button in the upper-right corner

of the Plate View to open a coincident display of the
plate’s results
Gen5 Secure Only: Sign a protocol or experiment

Buttons and Icons Guide | 53
Menu Tree Icons
Plate—not read: put the plate in the reader and click the
Read button
Plate read successful
Plate read paused by Stop/Resume step: when you’re

ready, put the plate in the reader, click the Read button,
and select Resume to continue
Plate read aborted: to begin again, put the plate in the

reader, click the Read button, and select Re-Read
Plate read in progress
Plate read error, which is always preceded by an error

message
Protocol
Procedure: define the reading parameters
Plate Layout: assign location of samples
Data Reduction: set up calculations
Runtime Prompts: define the information requested

when a plate is read
Patient Report Builder: select the content to print
Report Builder: select the content to print
File Export Builder: select the content to export

Menu Tree Icons
Power Export Builder: select the content to export to

Excel
RelPot Builder: select the content to export to the RelPot

application (available with the RelPot module)
Data Views: customize the appearance of data for online

viewing and reporting
Protocol Options: miscellaneous options for saving,

naming, exporting and calculating results
Plate Information: information obtained at runtime
Sample IDs: user-defined names or IDs assigned to

samples
Calculation Warning Log: Data Reduction-related errors

issued by unexpected curve or calculation results
Multi-Plate protocol view of data reduction statistics and

curves
Panel: multi-protocol experiment performed on one
plate
Paneled Protocols: lists the protocols run (or to be run)
in the panel experiment
Audit trail displays any logged events

Getting Technical Assistance | 55
Getting Technical Assistance
Gen5 is backed by a superior support staff. If the software fails to work

properly, please contact BioTek’s Technical Assistance Center (TAC). You can
call, write, fax, or email your questions and concerns to BioTek.
Email Support: tac@biotek.com
Fax Support
Send a fax with your questions or requests for help 24 hours a day:
• Technical Assistance Center (TAC): (802) 654-0638
• European Coordination Center: +49 (0) 7136.968.111
Phone Support:
You can telephone the Technical Assistance Center between 8:30 AM and 5:30
PM Eastern Standard Time (EST), Monday through Friday, excluding holidays.
Customer Service: (802) 655-4040
Technical Assistance Center:
• In the United States call: (800) 242-4685

• Outside the United States call: (802) 655-4740
European Coordination Center: +49 (0) 7136.9680
Essential Concepts
This section will give you a good basis of information for

understanding the structure and terminology of Gen5. You can find
more details and answers to specific questions by using the online
Help system. Select Help > Help Topics from the menu.
Experiment vs. Protocol ................................................. 58

About File Storage......................................................... 60
Best Practices ............................................................... 62
58 | Essential Concepts
Experiment vs. Protocol
Gen5 uses two common terms to define distinct elements of its toolkit. The
distinction is subtle and will have more or less importance depending on how
you use Gen5. In any case, you’ll work most efficiently by understanding each
role and making them work for you.
Protocol (*.prt) Experiment (*.xpt)
A protocol is a set of instructions An experiment has a copy of the

designed to capture, transform, and protocol and at least one plate. It
report and/or export data. executes the instructions provided by
the protocol to produce results.
Protocols are created and saved as Although an experiment is created

stand-alone files. They function as a using an existing protocol, that
template; an unlimited number of protocol can be modified within the
experiments can be based on one experiment.
protocol.
A protocol consists of reading Running an experiment is the only

requirements, like detection method way to process a protocol. The Gen5
and wavelength, and reading-related Quick Read function may at first
actions, like shaking and incubation appear to skip the protocol
(Procedure), plate layout, data development stage, but it uses the
reduction, and data viewing, default protocol and generally
reporting, and exporting parameters. requires reading parameters to be
defined
A protocol can be used repeatedly (as Multiple plates can be processed in an

is or modified) within experiments. experiment, each one considered a
By itself, a protocol does not produce unique assay with independently
results. Protocols do not have plates reported or exported results. The
associated with them. exception is multi-plate protocols,
described later.
.prt is the protocol’s file name .xpt is the experiment’s file name
extension. extension.
Experiment vs. Protocol | 59
Protocol (*.prt) Experiment (*.xpt)
A copy of the protocol is saved within An experiment is saved as the full

an experiment or as a stand alone .prt collection of procedures, formulas,
file. Since protocols do not have reporting definitions, and other
plates, they cannot generate data details, that is, the protocol and the
outside of an experiment. plate data from readings and
calculation results.
The Gen5 Secure level of software Data acquired and transformed in an

maintains an audit trail of all activity experiment is protected by an audit
and changes related to a protocol. All trail in both Gen5 Secure and other
other Gen5 software levels do not Gen5 software editions. The Reader
support this feature. Control edition does not support this
feature.
Changes made to a stand-alone Within an experiment, you can select

protocol are not reflected in any Save Protocol As to capture the
previously created experiments based current details of the protocol and save
on that protocol. A new experiment them as either a new protocol or as an
must be created to apply the revised overwrite of the original protocol.
protocol.
All newly created protocols and experiments (unless

another protocol is selected) are based upon the Default
Protocol.
Gen5 also supports more complex multi-plate protocols

that are not covered in this introductory material. Check
out Designing a Multi-Plate Protocol in the Gen5 Help
system.
About File Storage
File Types
Gen5 creates three file types:
• Protocol = .prt
• Experiment = .xpt
• Panel = .pnl
• The Gen5 executable file (.exe) and numerous other types of supporting
files, like a Microsoft Excel template, are also installed on the computer.
• In addition, Clarity Microplate Luminometer users will work with Clarity
protocol files, which use a .bpf extension. These files are referenced by
Gen5 files and they contain the reading parameters required to control the
luminometer.
Databases
Gen5 installs two databases on your system called LocalDB and SharedDB.
While the databases are always used for critical, internally used files, Gen5
offers you the choice of using the Windows File System or the Gen5 (SharedDB)
database for storing Gen5 Protocol (.prt) and Experiment (.xpt) files. This
option, combined with the ability to create multiple databases, allows you to
structure file storage according to your organization’s requirements.
• Files may be stored on the computer’s hard drive, on a network, or on a
CD or other portable medium. Windows Explorer or a similar application
can be used to view the file names and locations, and to move, copy,
rename, and delete files.
• Alternatively, protocol and experiment files may be stored in a secure,
shared-access database. This database, initially named SharedDB.mdb,
can be stored on a user’s computer or on a shared-access
network/computer (LAN). Gen5 provides a special file maintenance
utility for viewing the file names and their locations, and for moving,
copying, renaming, deleting, importing, and exporting files.
• Select the preferred method of storing protocol and experiment files at
System > Preferences > File Storage.
About File Storage | 61
File Location
During a typical installation:
• the program files are stored in this default location: C:\Program
Files\BioTek\Gen5 (software edition)
• the databases are stored in this default location:
• Windows XP and 2000 systems: C:\Documents and Settings\All
Users\Application Data\BioTek Instruments\Gen5 (software
• Windows Vista: C:\Program Data\BioTek \Gen5 (software
• Gen5 installs Protocol and Experiment folders in the respective File
Storage locations, for example, C:\Program Files\BioTek\Gen5 (software
edition)\Protocol.
When multiple user accounts share a computer, Windows Vista

may use its “VirtualStore” as the default location for file
storage. Talk to your system administrator about the best way
to manage file handling or use the Gen5 database to store
experiment and protocol files.
The databases are critical to Gen5 functionality. Make sure they

are not deleted from your system.
Best Practices
Like most software tools, Gen5 is flexible and offers several ways to accomplish
a task. Here are some recommendations for saving time and using it most
efficiently.
Efficiencies
• For an assay or experiment that you will run numerous times, develop a
Protocol to define the Procedure, Data Reduction, Data Views, and
Reports required. Then you can run an Experiment (File > New
Experiment) based on the Protocol whenever necessary. You can fine-
tune the protocol within an experiment, but remember to select File >
Save Protocol As to update the original protocol with your
improvements.
• Just as in word-processing documents, when you run similar types of
assays, you can use File > Save As to give you a head start creating a
new protocol based an existing one that contains the same or similar plate
layout, reading parameters, or other elements that will be repeated in
your new protocol.
• Define and customize Data Views before selecting what to include in
reports or export files. All the on-screen data (i.e., data views) can be
reported or exported. If you use on-screen views and paper reports
equally, it is most efficient to first fine-tune the Data Views, and then
include them in reports/exports.
• Always assign Blanks to the plate. Blanks can be deionized (DI) water,
buffer, reagent without analyte, substrate, and so on. When running
fluorescence cellular assays, a DI-water blank illustrates the background
contributed by the instrument and labware as separate from the cells and
media. Identify the location of the Blanks in the Plate Layout and Gen5
will automatically create the blank-subtraction data reductions.
• Back up your database regularly: BioTek recommends once per week for
most organizations. If you’re using a Gen5 Database for protocol and
experiment file storage, use the built-in Periodic Optimization feature.
Best Practices | 63
• Take action if you get a warning message about the remaining size of
your databases; see Maintaining Files in Gen5 Help for instructions on
archiving and deleting records.
• Turn off the Multi-Read Calculation option to improve Gen5 performance.
Calculation results will be the same, but your PC’s resources will not be
diverted for performing interim calculations. Find this option at Protocol
> Protocol Options > Calculation Options.
Time-Savers
• Partial Plate: for assays using strips or partially filled plates, especially if
the read steps are long or complicated, you can save time by telling the
reader exactly which adjacent wells or portion of the plate to read.
• Default Protocol: all newly created protocols and Quick Reads are based
on the Default Protocol. If some protocol elements, like read step, plate
layout, and report headers and footers, are largely the same for most of
your projects, you’ll save significant time by defining these elements
before creating the next protocol/experiment.
• Use the Gen5 automatic Save feature to create a new, date-stamped
folder for storing experiment records. This is an especially good practice
for large labs with multiple users who run hundreds of plates per day.
Gen5 will keep all the data organized by date. Define this kind of file
management setting in the Default Protocol so it will apply to all newly
created protocols.
Select Protocol > Protocol Options > Save Options.

• Print Preview: save time and paper by viewing reports on screen before
sending them to the printer.
Basic Tasks
This section provides instructions for some basic tasks.
Quick Read................................................................... 66
How to Create a Standard Curve ..................................... 67
Viewing Results............................................................. 68
Printing Results............................................................. 74
Testing the Reader ........................................................ 75
66 | Basic Tasks
Quick Read
In Gen5, a Quick Read is using the microplate reader connected to the PC to

read a plate and report the results. It’s called quick because it is accomplished
without taking the time to set up a protocol.
To perform a Quick Read:

1 Click Read a Plate from the Welcome page (and skip down to Step
4).
or click or select File > New Experiment.
2 Select Default Protocol.
3 Click (the Read button).

The Procedures dialog opens.
4 Click and enter the desired reading parameters and

any other needed steps. When you click OK to save and close the
Procedure, the Plate Reading dialog opens.
5 Click Read.
When the reading is done you can report the results. If you have a full-level
edition of Gen5 (Gen5, Gen5 ELISA, or Gen5 Secure) you can crunch the
numbers. See Set up Data Reduction Requirements for the Plate beginning on
page 84.
How to Create a Standard Curve | 67
How to Create a Standard Curve
Gen5 lets you create one or more standard curves for determining the
concentration of test samples:
1 Select File> New Protocol.
2 Select Procedure and define the Read step (and any other required
steps).
3 Select Plate Layout (see page 81):
• Define the Concentrations of the Standards.
• Assign the location of the standards, samples, and blanks (if
any) on the plate.
4 Select Data Reduction > Curve Analysis.
• Gen5 may have generated a “corrected” data set: if you
assigned blanks to the plate or selected Pathlength Correction
or Polarization in the Read step, you’ll want to select these
data sets for Data In for the Y-Axis Data to plot the curve.
5 On the Data In tab, use the drop-down to select the Y-Axis Data.
6 On the Curve Fit tab, choose a curve fit method.
7 Other options and requirements when defining multiple curves:
• Curve Name: replace the default “Curve” with a more
meaningful or unique name.
• On the Data Out tab, replace the default “Conc” for the Data
Set Name with a more meaningful or unique name.
• On the Data Out tab, define interpolations to plot on the
curve.
8 Define the reporting or export requirements and save the protocol.
Now you’re ready to run an experiment: click File > New
Experiment to read the plate and generate the curve. 0
68 | Basic Tasks
Viewing Results
You can instantly view the results of an experiment in the Gen5 main
workspace using the Plate View:
• After reading the plate (or otherwise acquiring data), in the Plate View
use the drop-down list for Data to display the raw data and any data
reduction results. You can also select Create new Matrix to define a
new view.
• Click the 3-dot button next to a data set to customize the view’s
appearance. This feature is also available in the Data Views dialog.
• Click the Quick Export button to instantly open the current view in
Microsoft Excel. Learn more about Gen5 Export Options on page 90.
• Asterisks (**) are used to signal a change: in the Gen5 title bar an asterisk
indicates the current file has been changed but not yet saved. When a data
set is enclosed by asterisks, it has been become invalid. Generally this is
because a Read step or Data Reduction step has been altered. Edit custom-
made data views to select valid data sets.
Viewing Results | 69
• 384- and 1536-well plates require resizing to effectively see the data.
Gen5 adds a button to the Plate View to zoom in on the top-left quadrant
of the plate and zoom out to view the entire plate. After zooming in, use
the scroll bars to bring the other quadrants into focus. Find more on
resizing the views in Gen5 Help.
• Multi-index readings offer another viewing option. Kinetic and scanning

reads generate views based on the number of intervals, wavelengths, or
positions defined. Use the spin buttons or enter the desired read index
and click Show to display it. Gen5 displays the time, wavelength, or
position of the selected read number.
• Kinetic and Scanning protocols can generate Well Analysis data sets
labeled Curves. In the Matrix tab, open the Curves data set and click on a
well for a Well Zoom. (384- and 1536-well plates show a magnifying glass
in the well in lieu of a curve.)
• Select the Statistics tab to view a table of data reduction results.
• Select the Graphs tab (when available) to view any standard curves.
• Select the Cutoffs tab or Validation tab (when available) to view the
values or results of the cutoff or validation formulas.
• Review the description of the Gen5 naming convention for the raw
data/results (next page).
L Important Notes:
• Gen5 may not display some data points by default; to see them you must
create your own Data Views. If you expected to see certain results that are
not currently displayed, try creating you own views.
• All data views are also available for Reporting and/or Exporting.
• Modify a data view to change the way results are reported, including the
number of decimal places and significant digits.
• Gen5 always uses your computer’s regional settings to display and input
data.
70 | Basic Tasks
Data Set Naming Convention

Gen5 names the data sets it creates as follows:
Name Meaning Example
wavelength Absorbance wavelength 450
wavelength[#] When two or more identical read steps 450[2]

are defined in the same Procedure, the
wavelengths are numbered
ex/bw & Fluorescence filter sets: 360/40, 580/40

em/bw excitation/bandwidth and
emission/bandwidth
em/bw Luminescence emission 460/40

wavelength/bandwidth
Lum Luminescence using Hole/empty Read 3:Lum

position in the filter wheel
Spectrum Spectrum scan Read 1:Spectrum
Curves [nm] Kinetic curves are automatically Curves [490]

generated during kinetic analysis; they
Curves Curves [360/40,
lead to Well Zooms
[ex/bw,em/bw] 460/40]
Read #:nm When more than one read step is Read 2:410
defined, the Read number names the
data set
Blank nm Blank well subtraction data set Blank Read

(created automatically by Gen5) 2:360/40
Corrected [nm] Pathlength corrected data set Corrected [405]

(Absorbance read option)
Polarization Data transformed per fluorescence Polarization

polarization formula
Name Meaning Example
Parallel Raw/blanked data from parallel Parallel Intensity

Intensity measurement
Perpendicular Raw/ blanked data from perpendicular Perpendicular

Intensity measurement x G Factor Intensity
ex/bw,em/bw Raw data from parallel or 485/20, 590/35

[Parallel] or perpendicular measurement [Parallel]
[Perpendicular]
485/20, 590/35
[Perpendicular]
Anisotropy Data transformed per anisotropy Anisotropy

formula
Data Reduction steps produce data sets named with the type of calculation and the
raw data set name. For example, Well Analysis steps could generate: Mean V
[340/40, 520/40] and Onset [Blank Read 2:490].
When a Step Label is entered for the Read or Data Reduction Step, the label is
included in the name of the data set. For example, a Step Label in an Absorbance
read step of Test1 would result in a raw data set of “Test1:450” and an
automatically generated blank-subtraction data set would be “Blank Test1:450”.
72 | Basic Tasks
Symbols and Special Notations

Depending on the current view, report, or exported text you may see any of the
various symbols and notations that Gen5 applies to call your attention to
special conditions.
Symbol Description
>(highest conc.) After fitting a standard curve and calculating the concentrations,
<(lowest conc.) Gen5 denotes wells falling outside the abscissa range with
>(upper limit concentration) or <(lower limit concentration);
<0.00 is displayed rather than negative concentrations, unless
it’s called in an interpolation.
????? ????? appears in a well in certain Matrix and Statistics views
when data for that well has not been obtained, cannot be
calculated, is based on out-of-range values, or has been failed
by the reader.
#### #### appears in a well when the size of the grid is not large
enough to show the value in the well. Resize the view to see the
contents. Or, change the Font used to display the value,
choosing a different “clipping” method.
[] In Well Zoom views when the original read range is reduced,
Gen5 plots the revised Calculation Zone with brackets: [ ]
*1234* Gen5 displays Masked values in data views and reports between
asterisks.
!1234! When Gen5 uses an out-of-range value in statistics calculations,
it is considered a biased result and will be reported between
exclamation points.
* Asterisks are displayed next to plate icons and in the title bar of
an experiment when a change is made or an action is taken but
the file has not yet been saved.
**dataset** Asterisks identify invalid data sets. This is generally caused by

changing the source Read step or Data Reduction step after the
data set was initially created.
#N/A Indicates an invalid data reduction item.
INJECT Synchronized Mode Procedures with multiple, appended kinetic

loops and one or more dispense steps use this symbol to
identify when dispensing occurred.
OVRFLW Measurements are not being collected, most likely due to

Symbol Description
inappropriate read parameters for the test specimen (overflow).
MISSED The reader did not capture a value for every Xenon flash
measurement taken for this well. The reader missed a
measurement and cannot report a reliable value.
Biased values may appear in a Statistics table when Calculation

Options are set to include out-of-range values. It is your
responsibility to determine whether the results are valid.
Well Analysis Results Table

When a Well Analysis Data Reduction step is defined, Gen5 shows the
calculation results in a table beneath the curve. The “Curves” or “Scan” data set
that leads to a well zoom must be the subject of a Well Analysis step; raw data
well zoom views do not include a Well Analysis Results table.
Calculation Zone
The range of reads considered for analysis is determined by the Calculation
Zone setting of the Well Analysis. When the original range is reduced, Gen5
plots the revised Calculation Zone with brackets: [ ]
Viewing Appended Kinetic Results

When one or more kinetic reads are appended, Gen5 combines them into one
data set. When a Dispense step occurs between the kinetic loops, Gen5
represents the event as a blue diamond on the X-axis timeline.
74 | Basic Tasks
Printing Results
Click the Print button to print the results of an experiment.
Prerequisite:
First, you must select the specific content for the report using the Gen5 Report
Builder (Protocol > Report Builder) or Patient Report Builder (Protocol
> Patient Report Builder).
Reporting in an experiment is done on a per plate basis:
• Highlight a in the menu tree and select Print/Print Preview.

• In a multi-plate experiment: You can select multiple plates by holding
the Ctrl key while highlighting them, or to select contiguous plates,
highlight the first plate, hold down the Shift key, and select the last plate.
Then, click Print.
Gen5 offers enormous flexibility in report output. After defining the report
elements, use the Print Preview option to view the report on screen before
printing it to paper. Unneeded columns and other individual report elements
can be removed or modified to improve the appearance and usefulness of the
report.
More Information:
In Gen5 Help you can find step-by-step instructions for creating and
customizing reports.
Basic rules and best practices for report designing are described in
Customizing Reports on page 86.
Testing the Reader | 75
Testing the Reader
Gen5 provides the following option for testing the reader.
Run a System Test

System > Diagnostics > Run System Test
The System Test for the Clarity Microplate Luminometer must

be performed using the Clarity PC software. Refer to the Clarity
Operator’s Manual for instructions.
Most BioTek readers perform a self-test every time they’re turned on, but when
you want to view and/or print the results of a system (aka optics) test:
1 Select System > Diagnostics > Run System Test.
When there is more than one reader attached to the PC, select the
desired reader and click OK.
2 When the test is completed:
• Fill in the text fields—User, Company, Comments—to be
included in the report of the test results. Then, click OK.
3 Print the report to retain a hard copy for your records.
4 Click Save As to convert the results to a text file. This is especially
useful when troubleshooting a reader. You can email the text file to
BioTek TAC. 0
Test History
Gen5 keeps the results of System Tests when they are performed using the
menu controls.
• To review or print the results, select System > Diagnostics > Test
History.
76 | Basic Tasks
Setting Up a Protocol
This section walks through the basic steps to creating a Protocol.

You’ll find more details in Gen5 Help. If you haven’t already done so,
read the difference between Experiments and Protocols in Gen5 in
the Essential Concepts section.
Design a Protocol .......................................................... 78

Defining the Reading Procedure....................................... 79
Defining the Plate Layout ............................................... 81
Setting Up Data Reduction.............................................. 84
Customizing Data Views, Reports, and Exports .................. 86
Building Reports............................................................ 87
Exporting Results .......................................................... 90
78 | Setting Up a Protocol
Design a Protocol
The menu tree provides a visual clue to the steps involved in creating most
protocols (File > New Protocol opens only the Protocol section of the menu
tree):
1 Define the procedure (beginning on the next page).
2 Define the plate layout (for all except Gen5 Reader Control software)
(page 81).
3 Define the Data Reduction requirements (for all except Gen5 Reader
Control) (page 84).
4 Define the runtime prompts to collect user input at runtime (plate
reading).
5 Define the reporting requirements (page 87).
6 Save the protocol.
Follow this sequence of tasks, when developing a protocol to

take advantage of the Gen5 automatically created data
reduction events. For example, when you add Blanks to the
Plate Layout, Gen5 automatically creates a Blank-Subtraction
data set.
You can find instructions for developing specific types of protocols in Gen5
Help. Or, you may want to begin with one of the Gen5 Sample Protocols (page
91).
Defining the Reading Procedure | 79
Defining the Reading Procedure
Protocol> Procedure
Set up the Procedure to control the reader: define the reading

parameters and related activities of the Protocol/Experiment.
Grayed out? Once a reading has been done in an experiment,

the Procedure cannot be changed for the current experiment. If
this isn’t the case, your System Administrator may have
restricted your ability to modify the protocol elements.
Grayed-out buttons mean the action cannot be performed by

the current reader or because previously defined steps, such as
a kinetic loop, limit the function.
How to:
1 Click a button to add that step to the procedure. Most buttons open a
screen for defining the parameters of that step, for example, Read lets
you define wavelengths.
When defining a kinetic or synchronized well/plate mode analysis,
add the Kinetic or Synchronized Mode steps first. Kinetic and
Synchronized Mode steps form a loop or block. Put the Read and
other valid steps to be performed inside the loop, between the Start
and End. Monitor Well is similar: first add the Monitor Well step and
then add a Read step inside the monitor-well loop.
2 Define the details of the step and click OK.
3 Select the Plate Type.
4 Click Validate to check the selection and sequence of the steps.
Defining the Procedure varies for Clarity Luminometers:

Gen5 opens the Clarity software to create or modify the
Procedure to run on the Clarity.
Features:
• You can drag and drop steps in the Procedure to change their sequence
order.
• Double-click a step to open it for editing.
• Highlight a step in the Procedure and then click an action button to add a
step before it.
• Select a step in the sequence and right-click for additional options.
• Click Validate at any time to verify the reader’s ability to perform the
current sequence of steps.
• Highlight a step and press Delete to remove it from the procedure.
Drag and drop is limited in Synchronized Modes. For example,

you cannot drag and drop a step into or out of a Well Mode
block.
Defining the Plate Layout | 81
Defining the Plate Layout
Protocol> Plate Layout

It’s easy to define the plate layout with the Gen5 tools for identifying samples,
standards, controls, and blanks. Follow these steps:
1 In the Well Settings box in the top-left corner, select the Type of
specimen.
2 Customize the ID or Well Identifiers, if necessary.
3 Define the Concentration or Dilution, if applicable.

4 Assign the well IDs to their corresponding locations in the plate grid
by clicking in the wells in the matrix. 0
• When you select a corresponding starting # the ID changes

accordingly for assignment to the plate.
• Use the Auto Select and Replicates options to speed up your work: set the
options and click and drag to fill multiple wells at once. Click a column or
row header to fill it.
The type of plate, for example, 96-well, is defined in the

Procedure and displayed in a representative matrix or grid
format in the Layout and Transformation screens.
L Helpful Hints:
• Set up your preferred default Well IDs in the Default Protocol. For
example, you can define PC (for Positive Control) instead of CTL1 for
Assay Controls. Well IDs defined in the Default Protocol are available
when defining the Plate Layout for all newly created protocols or
experiments.
• Use the Undo button at the bottom of the screen to undo the last action.
Up to 10 previous actions can be undone.
• To clear the grid and start over, right-click and select Empty Layout, or
to clear selected cells, set the Type of Well Settings to Empty and select
the cells you want to clear.
• You can print the plate layout. 384-well plates print out in two sections:
columns 1–12 and 13–24. 1536-well plates print in eight sections to fit all
48 columns and rows from A to Z and AA to AF.
• To copy the contents of the grid to a text file, right-click and select Copy
Layout. Open the text file in Microsoft Word or Notepad, right-click and
select Paste.
• Use the Dilutions Only/Concentrations Only button to apply only
dilution or concentration values (that you have previously defined) to
selected wells without altering the well’s identifier.
• When assigning concentrations/dilutions, well selection must be
compatible with the Replicate, Auto Select, and Filling option settings.
• You can resize the plate view in the standard Windows way: click and
drag the outer borders of the view, or click the maximize button in the
top-right corner. You can resize the rows and columns: hover your mouse
over a grid line between two numbered columns or alpha-labeled rows
until the cursor changes to a separator, then, click and drag.
• Each instance of a Sample and Sample Control Well ID, and each Assay
Control group can have a unique concentration/dilution value. Gen5
assigns a dilution index to the Well ID to keep track each instance.
Defining the Plate Layout | 83
• Gen5 maintains a unique list of concentration or dilution values for each

Well ID. To keep track of the values, Gen5 applies an indexing notation
when multiple dilutions or concentrations are applied to Assay Controls,
Samples, and Sample Controls:
IDi:n Well ID i = Well : n = dilution
ID Index index
For CTL 1 : 2
example
You’ll see CTL1:2 for the first assay control assigned
the second concentration value
SPL2:2 for the second sample assigned the
second dilution value
SPLC3:1 for the third sample control assigned
the first of multiple dilution or
concentration values
Setting Up Data Reduction
Protocol> Data Reduction

There are several options available for interpreting the results of your
experiment. Gen5 automatically creates the most commonly applied data
reduction steps.
Data Reduction Options:
• Transformation
• Curve Analysis
• Well Analysis
• Cutoffs
• Validation
• Fluorescence Polarization
Gen5 shows an invalid data reduction step by blocking out

its icon. Changing the Procedure—for example, wavelength or
sequence of steps, renaming a Read step, or making other
Protocol changes—can invalidate a data reduction step.
Generally, it is easiest to delete the invalid step and recreate it,
selecting valid options.
Top Six Things to Know about Data Reduction

1 Reductions are StepWise, that is, they can be built one upon another based
on their sequence in the Data Reduction dialog. For example, a data set
created in a previous step can be used in a later transformation, curve,
cutoff, and so on, if applicable.
Setting Up Data Reduction | 85
2 Gen5 creates four types of data reduction steps automatically:

• When blanks (BLK) are assigned to the plate, Gen5 automatically
creates a Blank Subtraction data set. Gen5 subtracts the mean of the
blanks from all other wells on the plate.
• A Well Analysis step is created when a multi-read index Read step is
defined: Kinetic, Spectrum, Area, or Linear Scan. If applicable, blank-
subtraction results are used to perform the well analysis.
• A Pathlength-Corrected data set is created when this option is selected
in an Absorbance read step.
• The Fluorescence Polarization (FP) transformation is performed
automatically when this reading mode is selected in the Read step. If
applicable, blank-subtraction results are used to perform the FP
reduction. In kinetic experiments, well analysis is based on the FP
transformation results.
3 Once the Data Reduction dialog is opened and saved, that is, OK is selected
to close it, Gen5-created reductions are no longer added, deleted, or
changed. Keep this in mind when modifying a protocol or experiment.
4 Plate-specific Data Reduction Variables can be collected from users when
they read the plate so Gen5 can use them in data reduction calculations.
First, define the variable in the Runtime Prompts, then write a formula
using the variable. At runtime, when the measurements and variables are
obtained, Gen5 performs the calculation using the input value.
5 Raw data sets used in Data Reduction steps are named according to the
Gen5 Data Set Naming Convention, which is based on the number of Read
steps defined in the Procedure. When the number of read steps is changed,
any previously defined Data Reduction steps are voided, because the data
set name is also changed. When you add or remove a Read step, you must
update the effected Data Reduction steps.
6 In the Data Reduction dialog, you can:0
• Drag and drop Data Reduction steps to change their sequence order.
• Select a step and right-click to delete it.
• Double-click an event to open it for modification or deeper review (or
right-click and select Edit).
Customizing Data Views, Reports, and Exports
Gen5 provides several tools for changing the appearance of views and reports.
All selections and customizations to views and to report and export elements
can be saved with the protocol (File > Save Protocol As), so they are
retained for all future experiments based on that protocol.
L Best Practices:
• A good habit to develop when setting up a Protocol is customizing the
content and format of the Data Views: the on-screen presentation of data.
The settings defined in Data Views become available for selection in the
Report and Export Builder screens, so it is most efficient to begin with
Data Views (after defining the Procedures, Plate Layout, and Data
Reduction details).
• Important: Attributes applied to data items and field groups in the Data
Views dialog take effect going forward. They do not replace, update, or
overwrite an item that has previously been assigned to a Report or Export
output. You must refresh the Report/Export contents after making any
changes to a data element, to capture them in the report. Any previously
saved Experiment will not reflect the content or formatting
changes.
• Gen5 limits the ability to customize system-provided views, but creating

new ones offers enormous flexibility. For example, if you do not use
Sample IDs, you’ll always have an empty Name column in the system-
provided Statistics tables. But, you can create your own view that
excludes the Name/Sample ID data point.
• You can choose the way Gen5 formats data/text that is too long to fit
completely in a field or column by Changing the Font for a data element.
• Field Groups (for use in reports) can be created only in the Data Views
dialog, but they can be modified in the Report and Export Builder dialogs.
Building Reports | 87
Building Reports
Protocol > Report Builder

Protocol > Patient Report Builder
About the Report Builder

Gen5 provides this tool to define exactly:
• what to include in the report
• how to format an item in the report
• where to place the item in the report
Define the Data Views and Default Protocol to speed up this step.
Report elements must be defined for each Protocol:

1 In the menu tree, under Protocol, select Report Builder or Patient
Report Builder.
2 Highlight Available Data Views and add them to the Report
Content/Content of each Patient section box.
• You can also drag and drop items into the Report
Content/Content of each Patient section box.
• Drag and drop to change the sequence order of items in the
Report Content/Content of each Patient section box.
• Edit an item in the Report Content/Content of each Patient
section box with a double-click or use the right-click menu.
• Highlight an item in the Report Content/Content of each
Patient section box and click Remove to deselect it (or use
the Delete key).
• To clear the slate and start over, click Remove All to deselect
all items.
• In multiple-read (multi-index read) protocols, like kinetic
analysis, it is necessary to select a range of reads or a range of
wells to include in a report. Certain data elements, like Well
Data, Well Results, and Well Zoom, require manual selection
of the specific wells to include. Except for area scan results,
you can select multiple wells for simultaneous reporting in
the resulting table or graph. For details see Reporting Well
Analysis Results in Gen5 Help.
Gen5 opens report items in Edit mode when a selection is required. For
example, when you select a Matrix item to report a kinetic read, you must select
a range of read numbers. One matrix will be reported for each read number.
3 Highlight an item in the Report Content/Content of each Patient

section box and click Page Break to add a page break to the report.
Gen5 inserts it just above the highlighted item. You can drag and drop
the Page Break to move its sequence order. (This step often works best
after you’ve read the plate and used Print Preview to assess the
layout of the report.)
4 Set up Headers and Footers, if desired. Find instructions in Gen5
Help. You may want to create them in the Default Protocol.
5 When the report is defined, in an experiment, click Print to

generate the report. 0
Items marked with asterisks (**) indicate one of three

conditions:
1. You must select specific data points from a very large (multi-
read) data set.
2. The experiment did not generate the expected results, or an
error occurred.
3. The Procedure and/or Data Reduction steps that generated
the item have been changed making it invalid.
Building Reports | 89
Click Print Preview to assess the report layout.
Using the Default Protocol

Using the controls in the Default Protocol, you can input text and select
protocol and experiment data to be included in all your reports. Your selections
can be modified, removed, or replaced in individual protocols without affecting
other experiments.
Content options in the Default Protocol may be limited by the

lack of a defined Procedure.
Important: Default Protocol settings are applied “going

forward;” they do not affect existing protocols. They are applied
only to newly created protocols.
System > Preferences > Default Protocol > Report Builder

Exporting Results
Gen5 provides these exporting tools:
• Quick Export: to instantly export the current view to a Microsoft

Excel worksheet
• Power Export: to export selected data to Microsoft Excel
• File Export: to export selected data, excluding curves, to a text file

(for use in another software application)
• Right-Click Menu Options—Copy to Clipboard and Save As: to
copy or save the current selection for use in another software
application
Prerequisites
For the Quick Export and Power Export features, you must have Microsoft
Excel 2000 or higher installed on your PC. Use the File Export or right-click
options if you do not have Excel.
About the Export Tools

• Power Export and File Export require selecting the content you want
in the output file before executing the export for a designated plate.
• You can save your export selections with the Protocol to reuse them
every time you run an experiment based on that protocol.
• Exporting data is like generating a report; it is done individually for
each plate. Although you can select the export content in a protocol,
you must run (or execute) the export in an Experiment (after selecting
one or more plates).
To preview the export-file output on screen before generating an export file, use the
Report Builder or Patient Report Builder and Print Preview: add the same elements
to a report as you do to the export, then click Print Preview to view the results
online.
Sample Protocols
Gen5 ships with numerous protocols so you can load one, customize
it to fit your particular requirements, and run an experiment.
Alternatively, we’ve provided step-by-step instructions for setting up
some common assays. Some are provided here; a longer list is
available in Gen5 Help.
How Do I Set Up My Assay?............................................ 93

Quantitative ELISA Example ........................................... 94
Dual Wavelength Absorbance.......................................... 98
Basic Fluorescence Assay Example................................... 101
Kinetic Fluorescence Assay Example ................................ 104
Fluorescence Polarization Example................................... 107
Max Binding Determination
Competitive Assay .................................................... 109
Toxicity/Cytotoxicity Assay ............................................. 113
Dispensing Reagent ....................................................... 117
92 | Sample Protocols
Numerous sample protocols are shipped with Gen5. You can use the protocols
to learn more about Gen5 and as a time-saver, customize them to meet your
needs and then run them in an experiment to obtain results.
Recommendation: Before making any modifications to the

sample protocols, open them, select File > Save As, and
assign them unique names. This will preserve the original
sample protocol for future use.
A matching experiment file is also shipped with Gen5 for use as a learning tool.
Many of the experiment files contain actual data so you can see how Gen5
presents the results on-screen and in reports.
Find the sample protocols and experiments shipped with Gen5 in the default
file storage locations. A folder for each detection method is available:
Absorbance, Fluorescence, and Luminescence. For Synergy 2/4 users, there is a
Synergy 2_Synergy 4 folder within each detection method folder.
• Gen5 Secure (and database users): Select File > Open Protocol, then in
the DB directory select the Samples folder. (Use the Up One Level
button.)
• All other levels of Gen5: Select File > Open Protocol and browse to
C:/Program Files/BioTek/Gen5/Samples.
• The Gen5 Welcome screen also offers the option to open a Sample File.
Important: The sample protocols must be considered as

examples provided for demonstration and guidance purposes. If
you plan to use these protocols or similar ones in a real
application, it is your responsibility to validate the protocol
parameters, including the report and export content (if
applicable), before using them.
Your system administrator can change the path and file names
described above. If you cannot find the Samples folder, contact
your system administrator.
Your reader may not support all of the sample protocols

provided. Review the descriptions in the Sample Protocols and
Experiments Guide to see if your reader is compatible with the
defined steps.
How Do I Set Up My Assay? | 93
Sample Protocols and Experiments Guide
You can review a complete description of samples in the Sample Protocols and
Experiments Guide shipped with Gen5: click the Start > All Programs >
Gen5 > Sample Protocols and Experiments Guide.
You can also find a summary listing and brief description of the sample
protocols in the Gen5 Help. Review the description of the sample protocol to
make sure it is compatible with your reader.
How Do I Set Up My Assay?
Here are step-by-step instructions for creating Gen5 Protocols to run common
assays. (More assay examples can be found in the Gen5 Help.) By following the
instructions and making some changes to wavelengths, names and other
details, you can adapt them for use in your lab.
• Quantitative ELISA Example on page 94
• Dual Wavelength Absorbance on page 98
• Basic Fluorescence Assay on page 101
• Kinetic Fluorescence Assay on page 104
• Fluorescence Polarization Example on page 107
• Max Binding Determination on page 107
• Toxicity/Cytotoxicity Assay on page 113
• Dispensing Reagent on page 117

Quantitative ELISA Example
To help you set up your own assay here is an example of the steps required to
run a quantitative ELISA assay. In this example we set up an endpoint
Absorbance read, subtract Blank wells from all others, plot a standard curve,
and define a Control to express the samples as a percentage of the control.
It may be easier to follow these instructions if you have already watched the
Gen5 Basic series of self-running tutorials (select Help > Tutorials).
To set up the protocol, we’ll define the:

• Reading Procedure
• Plate Layout
• Data Reductionso
Reporting results is the same process for all types of

experiments.
1. Defining the Reading Procedure

This assay example has the simplest read Procedure: a single-wavelength
Absorbance endpoint read.
1 Select File > New Protocol.
2 Select Protocol > Procedure.
3 Click Read and select the wavelength. Use the drop-down list or type
the wavelength in the text field (overwrite the current value).
4 Click OK twice to save the Procedure.
Quantitative ELISA Example | 95
2. Defining the Plate Layout

This step is critical for the data reduction steps to be defined later. Here are the
first seven columns of the plate layout we need:
The critical factor is using the Well IDs, not their location on the plate. We
selected the Type, defined the known concentration of the standards, and
assigned them to the plate.
Well ID Type Description
BLK Blank DI water only
STD Standard Known concentrations
CTL1 Assay Control Known Control
SPL Sample Unknown samples

3. Defining the Data Reduction Steps

Now that we’ve defined the reading parameters and plate layout, we can
define the data reduction steps: blank-well subtraction, standard curve, and
expressing samples as a percentage of the control. Gen5 creates the blank-
subtraction step for you automatically.
1 Select Protocol > Data Reduction.
Notice that one Transformation, named “Blank nnn,” where nnn is the
wavelength, has already been created. We’ll use the results of this
calculation to plot the standard curve.
2 Click Curve Analysis.
• Notice on the Data In tab, the Well ID is set to STD and X-
Axis Data to <Plate Layout Settings>. The known
concentrations entered for Standards are plotted on the X-
axis. Use the drop-down list for the Y Axis Data to select
Blank nnn (wavelength).
• Click the Curve Fit tab. Depending on your assay, you may
want to change the curve fit method to 4 Parameters or
another option, or use Log values on the X- or Y-axis. For
now, retain the defaults and click the Data Out tab. Take
note that the Data Set Name produced from the standard
curve is called Conc by default. (You can change it.)
• Click OK to save and close the curve.o
3 Click Transformation.
• For the Data In use the drop-down list to select Conc.
• Enter a New Data Set Name for the results of this calculation,
for example, %Control.
• In the Formula field enter: (X/CTL1)*100
Retain the default setting to Use single formula for all wells. X
represents the value of the current well. CTL1 is the well ID
for the control we assigned in Plate Layout.
Quantitative ELISA Example | 97
After the plate is read, you can return to the Data Reduction dialog to make
any needed changes, like the Curve Fit Method. Do not change the Data Out or
Data Set Names; this would invalidate the data reduction steps that use those
data sets.
4 Save the protocol.0

Now you’re ready to define your reporting requirements and run the protocol
in an experiment.
Dual Wavelength Absorbance
Here are step-by-step instructions for setting up a dual-wavelength absorbance read

with known concentrations of standards against which a linear regression curve is
plotted.
Create the Protocol

2 Double-click Procedure in the menu tree.

• Click Read to set the reading parameters. Keep the default
settings for Detection Method and Read Type: Absorbance
Endpoint.
• For Wavelengths, click the button for 2 and use the drop-
down list to select (or enter) the test and reference
wavelengths: 410 and 630 for this example.
• Click OK twice to close the Read Step, and then the Procedure
dialogs.o
3 Double-click Plate Layout to define the location of standards,

samples, and blanks on the microplate. For this example, the
standards are placed in the center of the plate. Modify the following
instructions to match the distribution of samples and standards on
your plate:
• Set the Well Settings Type to Standard and click the 3-dot
button next to the Conc. field to enter the expected
concentrations. For this example, leave 0 in the STD1 cell at
the top of the table. Select the Incr. checkbox, and enter 10 in
the field, then click in the STD1 cell, then in the STD2 cell, and
each subsequent cell in the table until STD8. Click OK to save
and close the concentrations.
• At the grid, set the Number of Replicates to 2, and select
Next Conc. under Auto Select. Click and hold as you roll the
Dual Wavelength Absorbance | 99
mouse over the 5 and 6 columns, (the cursor changes to a

black, down-facing arrow) to fill the entire columns.
• Set the Well Settings Type to Blank, keep the Number of
Replicates at 2, and click and drag over wells A1 and A2.
• Set the Well Settings Type to Sample, keep the Number of
Replicates at 2, and select Next ID under Auto Select. Click
and drag the cursor over the remaining wells in columns 1
and 2, and then 3–4, and then 7–12, to assign samples to all
the other wells of the plate.
4 Double-click Data Reduction.

Gen5 automatically creates the Blank-Subtraction transformations.
• Click Transformation to set up the calculation:
1 Click .
2 For DS1 (selected by default) use the drop-down list to select
Blank 410.
3 Select DS2 and use the drop-down list to select Blank 630.
4 Click OK to close the Multiple Data Set dialog.
5 For this example, call the New Data Set Name: Dual Wavelength.
Enter the name in the text box. Dual wavelength is also known as
Delta OD; you may want to use this name instead.
6 In the Plate Formula field enter DS1-DS2 to subtract the
reference wavelength (630) measurements from the test (410)
measurements. Retain the default: Use single formula for entire
plate.
7 Click OK to save and close the Transformation.
• Click Curve Analysis. In the Data In tab, use the drop-down
list to select Y Data: Dual Wavelength and click OK.
• Click OK to close the Data Reduction dialog.o
5 Set the Report parameters and Data Views as desired.

6 Save the protocol. Select File > Save and name it DualWave1 for this
example.0
Run the Protocol in an Experiment

Now you’re ready to run the DualWave1 protocol in an experiment.
1 Select File > New Experiment. By default, Gen5 highlights the
DualWave1 protocol in the dialog, making selection quick and easy.
2 If your plate(s) and the reader are set up, you’re ready to go: click
Read and follow the online prompts.0
Basic Fluorescence Assay Example | 101
Basic Fluorescence Assay Example
The following example of the steps required for nucleic acids quantitation
using a fluorescent stain, such as the dsDNA-specific PicoGreen, can help you
set up your own assay in Gen5.

This assay example defines a single-filter-set Fluorescent endpoint read.
3 Click Read and change the Detection Method to Fluorescence.
4 To set the Filter Set: filter-based reads use the drop-down list to select
the filter for Excitation and Emission; for this exercise, select 485/20
and 528/20, respectively. For monochromator-based reads, enter
485 and 528 in the text fields.
5 Keep the Optics Position set to Top and, if applicable, select a mirror,
such as Top 510, that corresponds to the selected filters. Top 50%
works with any filter.
6 Enter 65 for the Sensitivity setting when using filters, or enter 100
for the setting when using the monochromator.
7 Click OK twice to save and close the Procedure. 0

Define the plate layout in the usual way to reflect the arrangement of unknown
samples, standards, and blanks, if any, on the microplate. For the sample
protocol shipped with Gen5, the plate is set up as follows:
Defining the expected concentration of the standards is the required to plot a

curve.

With the reading parameters and plate layout defined, Data Reduction steps
can be created: a standard curve for determining the concentrations of the
unknown samples.
3 Notice on the Data In tab, the Well ID is set to STD and X Axis Data to
<Plate Layout Settings>. The known concentrations entered for
Standards are plotted on the X-axis. Use the drop-down list for the Y
Axis Data to select 485/20,528/20 (or 485,528).
4 Retain the default settings for Curve Fit and the Data Out tab. Take
note that the Data Set Name produced from the standard curve is
called Conc by default (you can change it). Click OK to save and close
the curve.
Basic Fluorescence Assay Example | 103
5 Click OK twice to save and close Data Reduction. 0
any needed changes, like the Curve Fit Method. Do not change the Data Out or
Data Set Names because that would invalidate the data reduction steps that use
those data sets.
4. Save the Protocol

1 Define the reporting requirements using the Report Builder, the
Patient Report Builder, or export options.
Now you can run it in an experiment: click File > New Experiment.
Kinetic Fluorescence Assay Example
To help you set up your own assay in Gen5 here is an example of the steps
required to measure antioxidant capacity based on the free radical damage to a
fluorescent probe, as in the Oxygen Radical Absorbance Capacity (ORAC)
Assays. This protocol deploys Gen5 Synchronized Plate Mode for precise
timing of the measurements.

This assay example defines a single-filter-set Fluorescent endpoint read.

3 Click Set Temperature and enter 37 for the temperature.
4 Click Plate Out/In and select Move plate in.
5 Click Delay and enter 0:30:00 to let the reader warm up.
6 Click Plate Out/In and select Move plate out.
7 Under Synchronized Modes, click Plate for a synchronized-plate
mode block.
8 Click Shake and change the Intensity to Fast and the Duration to
0:15.
Kinetic Fluorescence Assay Example | 105
9 Click Kinetic and set the Run Time to 1:00:00 and the Interval to
0:01:00.
10 Click Read and change the Detection Method to Fluorescence and
define the Filter Set. Filter-based reads use the drop-down list; for
monochromators enter the values in the text fields to set the Excitation
and Emission wavelengths, for this exercise, 485/20 and 528/20,
or 485,528, respectively.
11 For filter-based reads keep the Optics Position set to Top and select a
mirror, such as Top 510, if applicable. Top 50% works with any filter.
12 Enter 65 for the Sensitivity setting when using filters or enter 100 for
the setting when using the monochromator.
13 Click OK twice to close and save the Read step and the Procedure.

Define the plate layout in the usual way to reflect the arrangement of unknown
samples, standards, controls, and blanks on the microplate.
Defining the expected concentration of the standards is required to plot a curve.
Assigning blanks to remove background interference is always a good practice.
The sample ORAC protocol shipped with Gen5 also assigns

controls to the plate for assay validation purposes.

define the data reduction steps: two well analysis steps and a standard curve.
Gen5 automatically creates two steps: the Blank Subtraction
Transformation and Well Analysis for Max V.
2 Click Well Analysis to add another step to perform a calculation on
the results of individual read intervals.
• Enter a unique Label for this step, for example, AUC.
• Select Formula and enter it in the text field:

(R1/R1)+(R2/R1)+(R3/R1)+(R4/R1)+(R5/R1)+(R6/R1)+(R7/
R1)+(R8/R1)+...(R61/R1). This formula normalizes the AUC
(area under the curve); the results are used to plot the
standard curve to determine unknown concentrations.
• Click OK.
3 Click Curve Analysis to plot a standard curve:0
• On the Data In tab, select the Y-Axis Data. In this example,
choose the Well Analysis AUC: Formula Result [Blank
485/20, 528/20].
• Click OK twice to save and close the Curve and the Data
Reduction dialog.
any needed changes. Do not change the Data Out or Data Set Names because
that would invalidate the data reduction steps that use those data sets.

1 Define the reporting requirements using the Report Builder, Patient
Report Builder, or export options.
Now you can run it in an experiment: select File > New Experiment.
Fluorescence Polarization Example | 107
Fluorescence Polarization Example
To help you set up your own assay in Gen5 here is an example of the steps
required to perform a fluorescent polarization experiment.

This assay example defines a single-filter-set Fluorescent area scan read on four
wells.
3 Click Read and change the Detection Method to Fluorescence.
4 Select Use Filter Wheels, if applicable.
5 Select the Polarization checkbox.
6 Optionally, you can change the default settings for Light Source and
Read Speed.
7 To set the Filter Set, use the drop-down list to select the filter for
Excitation and Emission, for this exercise, 485/20 and 528/20,
respectively.
8 Gen5 automatically sets the Optics Position to Top and selects the
mirror in position 3 because the polarizers are located above this
mirror in the mirror holder. Since the mirror position is fixed for FP
analysis, you must select a filter set that corresponds to the mirror in
position 3.
9 Enter 65 for the Sensitivity setting.
10 Click OK twice to save and close the Procedure.

Define the plate layout to reflect the arrangement of unknown samples,
standards, and blanks, if any, on the microplate.

Gen5 automatically creates the Fluorescence Polarization steps, preceded by the
blank-subtraction transformations if Blanks were assigned to the plate layout.
You can add other Data Reduction steps as needed: Protocol > Data
Reduction.

1 Define the reporting requirements using the Report Builder, the
Patient Report Builder, or export options.
Now you can run it in an experiment: select File > New Experiment.
Competitive Assay | 109

Competitive Assay
To help you set up your own assay in Gen5, an example follows of the steps
required to run a competitive ELISA assay to determine maximum binding. In
this example we set up an endpoint Absorbance read, subtract Blank wells from
all others, subtract NSB (non-specific binding) wells from all others, plot a
standard curve, and define B/B0 as a percentage of bound sample and identify
the Total Activity (TA) wells.
To set up the protocol, we’ll define the:
1 Reading Procedure
2 Plate Layout
3 Data Reductions0

This assay example has a simple read Procedure: a single-wavelength

This step is critical for the data reduction steps to be defined later. Here’s the
plate layout we need:
The critical factor is using the Well IDs, not their location on the plate. We
customized the Well IDs for this example, changing the first three Assay
Control IDs to NSB, Bo, and TA. Then, we defined the known concentration of
the standards and assigned all of them to the plate.
BLK Blank DI water only
NSB Assay Control Assay-specific
Bo Assay Control Assay-specific
TA Assay Control Assay-specific
STD Standard Known concentrations

Competitive Assay | 111
SPL Sample Unknown samples

define the data reduction steps—blank-well subtraction, NSB subtraction—
determine the percentage bound, and plot a standard curve. Gen5 creates the
blank-subtraction step for you automatically.
Notice that one Transformation, named “Blank nnn” where nnn is the
wavelength, has already been created. We’ll use the results of this
calculation to build the next step.
1 For the Data In use the drop-down list to select Blank nnn.
2 Enter a New Data Set Name for the results of this calculation, for
example, B/Bo.
3 In the Formula field enter: (X-NSB)/(Bo-NSB)*100
1 Notice on the Data In tab, the Well ID is set to STD and X-Axis
Data to <Plate Layout Settings>. The known concentrations
entered for Standards are plotted on the X-axis. Use the drop-
down list for the Y Axis Data to select B/Bo (or whatever you
named the New Data Set Name in the previous step).
2 Click the Curve Fit tab. Depending on your assay, you may want
to change the curve fit method to 4 Parameters or another option,
or use Log values on the X- or Y-axis. For now, retain the defaults
and click the Data Out tab. Take note that the Data Set Name
produced from the standard curve is called Conc by default (you
can change it).
3 Click OK to save and close the curve.0
in an experiment.
More Advanced Options

• Monitor Wells: Sometimes it is necessary/desirable to wait until a certain
amount of activity has occurred in the plate before reading it. Gen5 will
periodically check designated wells until they’ve reached a certain
measurement when the Monitor Wells option is used. When the criterion
is met, Gen5 continues the Procedure, that is, the regular plate reading is
performed.
Toxicity/Cytotoxicity Assay | 113
Toxicity/Cytotoxicity Assay
An example follows of the steps required to run a Toxicity or Cytotoxicity assay

to determine LD50 (lethal dose). In this example the Read step is
straightforward, but the Plate Layout takes full advantage of Gen5’s ability to
customize Well IDs. We’ll create two samples with six dilutions each, and a
control and blank for each sample. Then, we’ll plot a standard curve based on
blank-subtraction and toxicity percentage. We’ll use the Gen5 Curve
Interpolation to identify LD50.

This assay example has a simple read Procedure: a single-wavelength
Alternatively, you may want to perform a kinetic analysis.

This step is critical for the data reduction steps to be defined later. Here’s the
plate layout we need:
The critical factor is setting up the Well IDs and dilution values. For the
Samples, (notice there are only two samples on the plate) we defined the
known dilution values and assigned them to the plate using the Auto Select
features Next ID and Next Dil for 3 Replicates. We customized the Well IDs for
the Sample Control IDs to BLK to have sample-specific blanks and assigned
them to plate without using the Auto Select features. Assay Controls were
assigned to the plate in the same way as the BLK1 and BLK2.
Toxicity/Cytotoxicity Assay | 115
SPL Sample Unknown samples with

dilution values
CTL Assay Control Sample-specific
BLK Sample Control Customized to be sample

specific

define the data reduction steps: blank-well subtraction, toxicity percentage
determination, and a standard curve.
1 For the Data In only one data set, the raw data from the Read step,
is available.
2 Enter a New Data Set Name for the results of this calculation, for
example, %Toxicity.
3 Deselect the Use single formula for all wells.

4 In the Current Formula field enter: (X-BLK1)/(CTL1-
BLK1)*100
5 Click and drag over the SPL1 wells to assign the formula to them.
6 Change the Current Formula for SPL2 wells, (X-BLK2)/(CTL2-
BLK2)*100, and assign the formula to them.
7 Click OK to save and close the Transformation.
1 On the Data In tab, the Well ID is set to SPL- All IDs and X-
Axis Data to <Plate Layout Settings>. The known dilutions
entered for Samples are plotted on the X-axis. Use the drop-down
list for the Y Axis Data to select % Toxicity (or whatever you
named the New Data Set Name in the previous step).
2 Click the Curve Fit tab and change the curve fit method to 4
Parameters (unless you prefer another method).
3 Click the Data Out tab, in the Interpolations table enter 50 (and
90, if desired). 50 represents 50% toxicity and it will be plotted on
the curve.
4 Click OK to save and close the curve.0
4 Save the Data Reduction steps (click OK).
in an experiment.
Dispensing Reagent | 117
Dispensing Reagent
Dispensing reagent during an experiment is affected by the type of analysis:

• Dispensing Reagent in a Kinetic Analysis (below)
• Dispensing Reagent in an Endpoint Analysis on page 119
Dispensing Reagent in Kinetic Protocols

Reagent can be dispensed during a kinetic read either manually or using a
reader with injectors.
Manually Dispensing Reagent

Create a protocol with an extended kinetic interval; add enough time to
dispense reagent to the plate between readings. During an interval, when the
reader is idle:
1 Push the carrier eject button on the front of the reader to eject the
plate.
2 Perform the dispense using a pipette.
3 Push the carrier eject button to draw the plate back in.
4 The read will continue as scheduled.
Readers with Injectors
Standard Mode
Define two kinetic read steps around a dispense step in the Procedures:
1 Define the Kinetic timelines and the Reading parameters of the first
loop.
2 After the End Kinetic step, add a Dispense step to dispense the
reagent. (You can add a Delay and/or Shake step after dispensing and
before the next kinetic loop.)
3 Add a second Kinetic loop. Kinetic settings can differ. Add a Read
step to the loop, and check Append to Previous Kinetic Data. Selecting
this option copies the previous defined reading parameters, for
example, wavelength/filter set, to this step.0
Synchronized Mode
For Fluorescence or Luminescence analysis select Plate or Well Mode to most
precisely control the timing of your experiment. Learn about Synchronized
Modes in Gen5 Help.
1 Add a Plate or Well mode block to the Procedure.
2 Create a Dispense step to dispense the reagent. (You can add a Delay
and, if supported by the reader, a Shake step, after dispensing and
before reading.)
Dispensing Reagent | 119
3 Add a Read step to the block, defining the reading parameters as

needed.
4 Within the block you can repeat any or all of the options: Dispense,
Delay, Shake in Plate mode, and Read, again. Note: the first Read step
in the block sets the parameters.0
Dispensing Reagent in Endpoint Analysis
Manually Dispensing Reagent

Add a Plate In/Out step to the Procedure:
1 You might want to incubate the plate before adding the reagent. If so,
and your reader is capable, add a Set Temperature step.
2 Add a Plate In/Out step. Enter Add Reagent in the Comment field.
3 Add a Read step.0
Readers with Injectors

Define a dispense step in the Procedure:
1 Create a Dispense step to dispense the reagent.
2 Add a Delay and/or Shake step after dispensing, if desired.
3 Add a Read step.0
L Other Options
Multi-Detection Methods
For readers capable of performing multiple detection methods, Gen5 supports
multi-detection kinetic protocols.
Discontinuous Kinetic Procedure
A Discontinuous Kinetic Procedure can be defined to execute a sequence of
readings over an extended time period. Use the Procedure’s Advanced Options
to conduct an experiment that requires long periods of downtime (for rest or
incubation) between reads. Learn more in the Gen5 Help.
Troubleshooting
This section provides guidelines for resolving errors, like

communication errors between the reader and the computer, and
unexpected or poor performance. You can find more troubleshooting
tips in the Gen5 Help.
Troubleshooting ............................................................ 122

Communication Errors ................................................... 123
Restoring Optimal Performance ....................................... 125
Fluorescence/Luminescence Measurements....................... 126
System Administrator’s Password .................................... 130
Database Errors ............................................................ 131
122 | Troubleshooting
Troubleshooting
Here are some guidelines for error recovery:

• First Response: Running a System Test is the best first response to a
reader error. The test may restore the reader’s initial settings and
computer communication capability. Note: to stop the alarm on readers
without keypads press the plate-carrier button.
• Reboot your Computer and Reader: When you can’t run a system test,
for example, Gen5 is not responding, or when running a system test
doesn’t resolve the issue, turn off your computer and reader, check all the
cabling (i.e., make sure your serial or USB cable is in good condition and
is properly connected to the PC and reader), and then, power on your
computer and reader. These steps should refresh the devices and reset
communication parameters.
Visit BioTek’s website for useful suggestions on getting the most from your
reader: www.biotek.com/products/technotes.php
• Other Known Issues: Gen5 installs a Read Me file in the root directory;
the default path is C:\Program Files\BioTek\Gen5 (software
level)\ReadMe.txt. It lists known issues you may have encountered.
Locate and review the file and contact BioTek TAC for additional
information or support.
• Synergy 2/4 to non-Synergy 2/4 readers (and vice versa): Protocols
created with a Synergy 2 or Synergy 4 reader are not instantly compatible
with other readers and vice versa. To correct the error “Procedure was
defined for a different reader,” open the Procedure and click Validate. If
this does not correct the error, open each step in the Procedure and review
it for compatibility with the current reader.
• Windows Vista Missing Files: The Gen5 installation routine attempts to
avert potential file sharing issues on Vista, but when multiple users share
a computer, Vista may use its “VirtualStore” as the default location for file
storage.
Communication Errors | 123
Communication Errors
FYI
• To prevent damage to the reader, always turn off the reader or the
computer before removing or inserting a communications (serial or USB)
cable.
• Gen5 and the reader-communication parameters supersede the Windows
settings. Windows communication port configuration settings should not
need adjustment to enable proper communications.
When the computer (PC) won’t communicate with the reader

• Confirm that the reader passes its system self-test. All BioTek
readers perform a self-test when turned on. Refer to the reader’s
operator’s manual for more details. The reader will not communicate if it
fails an internal system test. Non-keypad readers beep continuously when
the system test fails. (Press the plate-carrier button to stop the alarm.)
Keypad readers display an error message when the test fails. Refer to the
operator’s manual to resolve the failure or contact BioTek TAC.
• Make sure the serial or USB cable is in perfect condition and
properly attached to the port defined in the Reader Configuration dialog
(e.g., COM 1). Correct and reboot both PC and Reader. Test
communication.
• Confirm the Baud rate (or transmission speed) defined in the Gen5
Reader Configuration matches the reader’s settings. Consult your reader’s
operator’s manual for the correct rate. Correct the Gen5 Reader Settings to
match the reader and reboot both PC and Reader. Test communication.
• Disable the Calculation Option: Perform data reduction after each
read to give Gen5 sufficient time between obtaining measurements to
perform calculations.
• Confirm that the serial/USB cable was obtained from BioTek.
Serial/USB cables are not universal. Consult the reader’s operator’s
manual for proper cable configuration or contact BioTek customer service
to purchase a factory-tested cable. After installing a known good cable,

reboot both PC and Reader. Test communication.
• Confirm with your computer supplier or a local PC technician that the
serial port has been enabled. For example, the IBM® Thinkpad® was
originally shipped with the serial port disabled. Correct and reboot both
PC and Reader. Test communication.
• For advanced PC users, the serial port of the reader and PC can be
independently tested using an independent serial-communication
software package such as Windows Terminal, Hyper Terminal, or
ProCom. BioTek does not support or sell these programs. •
• Select flow control for “XON/XOFF” and send an ASCII asterisk
symbol (*) to the reader. The reader should initiate a self-test and
return the results to the PC. If the reader fails to communicate, and
Steps 1 through 5 do not resolve the problem, test the reader on an
alternative PC to confirm which device is at fault. Please contact
BioTek if the reader is diagnosed to be faulty.
Restoring Optimal Performance | 125
Restoring Optimal Performance
Numerous factors can affect your computer’s performance. If you notice a

slowdown in Gen5’s performance, follow these suggestions:
• Close all other applications, including Internet browsers, when running
Gen5.
• Do not display Gen5’s Curves data in the Plate View while performing a
kinetic analysis. Wait until the read step is finished before viewing the
Curves data set. Displaying the Curves data set during a Kinetic read can
consume excessive resources resulting in performance degradation. You
can drill down to a Well Zoom to monitor the progress of one well, then,
leaving the Well Zoom open, change the Matrix Data to a numeric view.
• Disable the Calculation Option “Perform data reduction after each read”
to give Gen5 sufficient time between obtaining measurements to perform
calculations. Select Protocol > Protocol Options > Calculation
Options.
• Disable the auto-Save option: Save when: Interim read completed. Change
the Save Options to free up resources. Select Protocol > Protocol
Options > Save Options.
Fluorescence/Luminescence Measurements
Here’s a list of potential problems, the possible cause, and a remedy:
Fluorescence/Luminescence Readings Too Low

• Possible cause: The Sensitivity in the Read Step dialog is currently
set too low
Raise the Sensitivity to an appropriate level. For fluorescence, the
Sensitivity is usually set between 45 and 130. For luminescence it is
usually set between 100 and 200. Learn more in the Gen5 Help.
If using Automatic Sensitivity Adjustment, use the Scale to High Well
option and set the target value to be between 20,000 and 80,000 for
standard range, or 1,000,000–3,500,000 for extended range.
• Possible cause: The wrong filters are selected in the Read Step
dialog
Examine the current filter settings and make any corrections. If the
filter settings appear to be correct, check the locations of the actual
filters in the instrument.
• Possible cause: Top probe is too high
• Synergy HT: If a plate cover is not being used, lower the top
probe to 1 mm above the selected plate using the Top Probe
Vertical Offset option in the Read Step dialog.
• Synergy 2/4/Mx: Gen5 generally positions the top probe at
the optimal height for fluorescence reads; it focuses the beam
above the well. Refer to Gen5 Help and use the Top Probe
Vertical Offset option in the Read Step dialog to make
adjustments.
• FLx800: Refer to the operator’s manual for instructions to
manually lower the Top Optical Probe.
Fluorescence Background Too High

• Possible cause: Using incorrect microplates
Solid black plates and top probe reading lower the background. Black
plates with clear bottoms lower the background if bottom reading is
necessary. Corning 3615 or 3614 (for cell culture) are appropriate.
Fluorescence/Luminescence Measurements | 127
dialog
• Possible cause: Phenol red is used in the media when exciting at
485 nm and reading at 528–530 nm
Eliminate or replace the phenol red.
• Possible cause: Cells, media and other contents fluoresce
Use deionized-water blank wells as a diagnostic tool. The blank-well
reading will help you determine the background value contributed
by the instrument, labware, and media.
• Possible cause: The top and/or bottom probe needs cleaning
Refer to the operator’s manual for instructions to open and clean the
reader’s internal components.
• Possible cause: The instrument has fluorescing material spilled
inside
Refer to the operator’s manual for instructions to open and clean the
reader’s internal components.
• Possible cause: The Sensitivity in the Reading parameters dialog is
currently set too high
Lower the Sensitivity setting. The background should still read
higher than zero. 200 is a recommended minimum and a value of
1000 takes advantage of the system’s five-digit resolution.
Reader Not Achieving Desired Fluorescence Detection Limit

dialog
• Possible cause: Using incorrect microplates
Solid black plates and top probe reading lower the background. Black
plates with clear bottoms lower the background if bottom reading is
necessary. Corning 3615 or 3614 (for cell culture) are appropriate.
• Possible cause: The Sensitivity is currently set too low

Raise the Sensitivity setting until the background wells read at least
200 RFU (1000 RFU is preferred).
• Possible cause: Readings are taken using the bottom probe
Switch to the top probe (Optics Position in the Read Step dialog).
• Possible cause: The solution volume is 50 ul or less
Increase the solution volume to 150–200 ul, if possible.
• Possible cause: Wrong pH
Fluorescence is very pH dependent. Use the appropriate pH.
• Possible cause: Phenol red is used in the media when exciting at
485 nm and reading at 528–530 nm
Eliminate or replace the phenol red.
• Possible cause: Top probe is too high
• Synergy HT: If a plate cover is not being used, lower the top
probe to 1 mm above the selected plate using the Top Probe
Vertical Offset option in the Read Step dialog.
• Synergy 2/4/Mx filter-based reads: Gen5 generally

positions the top probe at the optimal height for fluorescence
reads; it focuses the beam above the well. Refer to Gen5 Help
and use the Top Probe Vertical Offset option in the Read
Step dialog to make adjustments.
• Synergy 4/Mx mono-based reads: Gen5 positions the top
probe at 4 mm for 96-well plates and at 6 mm for 384-well
plates because these settings were determined to be the
optimal height. You may be able to lower the probe, but
lowering it may simultaneously require a small adjustment to
the Column Offset, as well.
• FLx800: Refer to the operator’s manual for instructions to
manually lower the Top Optical Probe.
• Possible cause: Transfection efficiency in gene expression is too
low
Use more cells, or improve the transfection efficiency.
• Possible cause: DNA is old or of poor quality
Use high quality, new DNA.
Fluorescence/Luminescence Measurements | 129
• Possible cause: Not using nuclease-free buffer in DNA quantitation

Use nuclease-free buffer.
• Possible cause: Poor dilution methods
Use appropriate dilution method in tubes.
Reader Over-ranging in Fluorescence

• Possible cause: The Sensitivity in the Read Step dialog is currently
set too high
Lower the Sensitivity setting. If using Automatic Sensitivity
Adjustment, try the Scale to High Well option and set the High Value
in the range of 50,000 to 70,000.
Bandwidth Verification Failed

• Error or warning messages are issued when Gen5 detects
overlapping wavelengths or bandwidth
Select/enter Filter Set wavelengths that do not overlap. Learn more
about Gen5 bandwidth verification in Gen5 Help.
Error during Auto-Sensitivity Determination

• Reader cannot fulfill request to determine optimal Sensitivity
Gen5 displays an error message when the reader cannot determine
the optimal sensitivity based on the defined reading parameters.
• Luminescence integration time in the Synergy 2/4 should be <= 1 sec and
> 1 ms, especially when scaling to low wells.
• Manually enter a Sensitivity value or use an alternative method to
determine the optimal sensitivity, if the error persists. Learn more in Gen5
Help.
System Administrator’s Password
Contact BioTek Customer Care if you’ve lost or forgotten the System

Administrator’s password.
The System Administrator’s password does not expire, but if a change in
personnel or some other cause has resulted in your team not knowing the
password, you can contact BioTek for a new one.
Gen5 ships with the System Administrator’s password set to admin.
Database Errors | 131
Database Errors
Certain conditions can cause database-related errors:

• The database file is not available. Potential causes: the shared
database is on a network and the cable is unplugged, or the file is
locked by another user.
• The database file is corrupt. Potential cause: an incomplete write
operation occurred because Gen5 was closed unexpectedly due to a
power outage or hardware problem.
• Verification of the database capacity failed. Potential cause: the
maximum size of the database (2 gigabytes) has been exceeded.
• File importation failure. Potential causes: the file name extension is
wrong; a protocol file was misnamed with an .xpt filename extension
instead of .prt.
Fixing the Errors

1. First, make sure non-Gen5-system issues are resolved, for example:
• Network cables are plugged in and the network is up and
running.
• Another Gen5 user is not currently performing database
maintenance routines.
• The database configuration for both the SharedDB and
LocalDB databases point to the expected locations.
2. Then, follow these instructions for fixing the errors:
• Fixing a Database Connection Error (page 132)
• Fixing a Corrupted File Error (page 133)
• Fixing a Database Capacity Error (page 133)
• Fixing a File Importation Error (page 134)
3. If the above solutions do not work, try restoring an archived database
(page 135).
Fixing a Database Connection Error
Connection to SharedDB failed

This type of error is most commonly caused by a network timeout or
disconnection, or corruption to the SharedDB file residing on a
network.
To fix:
1 Make sure you are properly connected to your network, if applicable,
that is, cables/wires are installed and the network server is live.
2 Click Reset Connection at the Database Configuration screen.
Gen5 immediately opens the Database Configuration screen after
displaying the error message. Otherwise, select System >
Database Configuration.
Important: Click Reset several times, if needed, until it is

grayed out.
Quit: If Gen5 cannot restore the network connection, usually

because of external factors—for example, the network is down—
when you click Quit at a secondary error message, Gen5 will
save any currently opened and modified files to the Windows
Temp directory. When the database is restored you can import
the files from the Temp folder to the database.
3 When the database connection has been repaired, click Optimize.
If Reset button fails:

If your network is performing as expected, and you’ve checked the cabling
from your PC to the network, and the Reset button fails to re-establish a
connection with the shared database:
1 Reboot your PC.
If the same message is displayed:
2 Restore a previous version of the database (page 135) or contact your
System Administrator to inspect and repair your system connection.0
Fixing a Corrupted File Error
Database file is corrupt
There are two potential ways to fix a corruption error:

• Reboot your PC to try to clear the error by restarting the system.
• Install an archived version of the LocalDB.mdb: find instructions to
restore a backup of the database on page 135.
Windows Explorer provides an option to hide certain folders

from view. If you cannot find the Application Data folder
(C:\Documents and Settings\All Users\Application Data) it is
probably hidden. In Explorer, select Tools > Folder Options
> View and enable Show hidden files and folders.
If these options fail to repair Gen5’s behavior, contact BioTek

TAC.
Fixing a Database Capacity Error
When the cumulative size of the files in your database nears or exceeds its
capacity (2 gigabytes) Gen5 displays an error message or warning. You must
reduce the size of the database. There are numerous ways to accomplish this;
the least invasive method, to remove files, is described here.
Prerequisite
the Administrator (System > Administrator Login) to access these
controls.
Optimize the database before proceeding to see if Gen5 can

compact the database sufficiently to comply with its size
limits. Select System > Database Configuration >
Optimize. When the process is finished click Stats to check
the current size.
To reduce the size of your database:

1 Select System > Maintain Files.
2 Highlight multiple database records; hold down the Ctrl key while
selecting records.
3 Right-click and select Export to Disk from the pop-up menu.
4 Select a folder (using the standard Windows dialog) where you want
to save or archive the Gen5 records.
5 In the Maintain Files dialog, highlight the same records, and click
Delete (or right-click and select Delete).

6 Finally, optimize the database to ensure it is running in top form. 0
Fixing a File Importation Error
File importation failure

The requested file may be a Protocol file
The requested file may be an Experiment file
Gen5 tests the file’s format when you perform certain operations like importing
a file and File > Open. One of these error messages may be displayed when
an incompatibility is found.
Change the filename extension to fix these errors
1 Locate the offending file: depending on the current File Storage mode:
• Using the Gen5 Database, select System > Maintain Files.
• Using the Windows File System, use Windows Explorer.
2 Highlight the file, right-click, and select Rename:
• Change the filename extension for a misnamed Protocol file
from .xpt to .prt.
• Change the filename extension for a misnamed Experiment
file from .prt to .xpt.
3 Retry the desired action, for example, open the file or import the file.
Restore an Archived Database
When the Gen5 error recovery processes cannot resolve database errors, a final-
resort solution is to replace the current database file with an archived or backup
copy of it. Ideally, you or your System Administrator has regularly (or at least
recently) backed up or archived the database. The Gen5 Optimize tools can
perform backups regularly.
Check the Auto-Optimize Settings, if they were defined, to determine the

location of the last-saved backup file.
How to
1 Using Windows Explorer, locate the last-saved archived or backed-up
database file.
2 Copy and paste it to the desired location. Rename the database file,
too. (All of these commands are available from the right-click menu.)
3 In Gen5, select System > Database Configuration.
4 Select the SharedDB tab.
5 Next to the Source field, click the 3-dot button and browse to the
location selected in Step 2.
6 Click OK. 0
Index
! Automatic Sensitivity Adjustment

Fluorescence/Luminescence
!exclamation points! ......................72 .......................................... 126
Automatically generated data
* reduction.................................... 84
* Asterisks explained.....................68
B
. Backup files .......................... 132, 133
.prt..............................................58, 60 Bandwidth Verification .............. 126
.xpt .............................................58, 60 Bandwidth Verification Failed .. 126
Basics............................................... 42
? BioTek’s Technical Assistance
Center......................................... 56
????? .................................................72
Blank Read1 ................................... 70
[ Blanks
Subtracting blank wells........ 84
[brackets].........................................72
Build
Report ..................................... 87
A
Buttons
A260/A280 .....................................78 explained................................ 52
About
Data Reductions ....................84 C
the Menu Tree........................51 Calculation Zone
Acquiring data ...............................84 Symbols .................................. 72
Add Calibrator/Standards Plate
New User................................30 Protocol ...................................... 78
Administrator, Power User ..........29 Changing
138 | Index
Font .........................................86 Data Views ..................................... 68

On-screen appearance ..........86 Database Management
Clarity Database Utilities .......... 24, 131
Setup .......................................16 Error recovery ..................... 131
Common Application Data Folder Reduce the size.................... 133
.....................................................25 Default data reduction.................. 84
Communication errors ................123 Defining
Concentrations Procedures ............................. 79
Determine by Curve Fit........67 Reporting requirements....... 87
Connecting a Reader ...............14, 18 Timelines.............................. 101
Copying Design
Gen5Shared Database...........22 Export ..................................... 90
Corrected.........................................70 Protocol .................................. 78
Creating Designating a System
New Protocol .........................78 Administrator ........................... 22
New User Accounts ..............22 Dispense reagent
Standard Curve .....................67 in a Kinetic Analysis........... 117
Curve in an Endpoint Analysis .... 119
Standard .................................67 Display measurements ................. 68
Curve Analysis...............................84 DNA Purity .................................. 126
Customizing Dual Wavelength
Data Views .............................86 Data Reduction...................... 84
D E
Data Files Email Notification feature
File Storage.......................20, 60 configuring ............................ 37
Organizing your Files ...........26 defining a template............... 38
Data Reductions Email Support ................................ 56
How to set up.........................84
Error Messages
Top 5 things to know ............84 Communication Errors ...... 123
Data Reductions.............................84 Database Errors................... 131
Data Set Naming Convention ......70 Excel
Data Views Shortcuts not working........ 122
Customizing...........................86 Experiment
| 139
printing results ......................74 H

Experiment vs Protocol.................58
Help>About Gen5 ......................... 56
Export Builder ................................86
How to
Exporting Create a standard curve . 67
Results.....................................90
View results ..................... 68
F Print results...................... 74
File locations...................................60 Set up Data Reduction.... 84
File Management ...........................26 Set up Kinetic/Time course
File Storage ...............................20, 60 analysis ...................... 101
File types .........................................60 Set up kinetic analysis .. 101
Fluorescence/Luminescence
Background too high ..........126 I
Readings too low.................126
Icons
Troubleshooting explained................................ 52
measurements.................126
Improve the transfection ............ 126
FLx800 .............................................14
Initial Setup .................................... 18
Font
Initial Setup Tasks ......................... 23
Changing ................................86
INJECT............................................ 72
Font ..................................................86
Injectors......................................... 117
G Install Gen5 Secure........................ 23
Installing Gen5............................... 18
Gen5 Secure Only ..........................29
Instrument Not Achieving Desired
Gen5 Secure Series.........................22
Fluorescence Detection Limit 126
Gen5Shared Database
Instrument Over-ranging ........... 126
Copying ..................................22
Introduction ................................... 42
Gen5Shared Database ...................22
Getting Technical Assistance .......56 K
Getting to Know Gen5 ..................42
Kinetic Absorbance Assays.......... 78
Glow Luminescence ......................78
Kinetic Analysis........................... 101
Graph...............................................86
Kinetic Fluorescence ................... 101
Groups.............................................29
Kinetic reads
How to create a protocol.... 101
140 | Index
L O
LD50 ................................................78 Optics Position............................. 126
Learn About Out-of-range values ...................... 72
Buttons....................................42 Overflow (OVRFLW).................... 72
Gen5 Databases .....................24
Login P
changing another user’s
Page Break
password ...........................30
add to report.......................... 87
controls/parameters .............32
Page Break ...................................... 87
Low UV Applications....................78
Password
Lowry Protein Assay.....................78 expired.................................... 34
Lum..................................................70 Pathlength Correction................... 84
Luminescence Perform
Troubleshooting ..................126 Kinetic analysis ................... 101
Multiple detection methods
M .......................................... 101
Matrix ..............................................68 Quick Read ............................ 66
Menu Tree.......................................51 Performance degradation........... 122
Microplate Permissions .............................. 21, 29
Layout samples......................81 Plate Reading ................................. 66
Quick Read.............................66 Plate#
Microsoft Excel 2000......................90 Viewing results ..................... 68
Microsoft Word ..............................90 Plate, Reading a Partial................. 79
Minimum Requirements.................4 Power User ..................................... 29
Missing Files.................................122 Preferences
Multi-index reads ..........................68 Startup.................................... 30
Print Preview ................................. 87
N Printing
Defining the Report .............. 87
Naming
Data Sets .................................70 Quick Print............................. 74
New User Accounts.......................30 Printing ........................................... 74
Printing ........................................... 87
Procedure ....................................... 79
| 141
Procedure, set up ...........................79 Reports............................................ 87

Protein Quantitation......................78 Requirements for plotting a curve
Protocol vs Experiment.................58 ..................................................... 67
Protocol’s Procedure .....................79 Results
Meaning of symbols, asterisks
Q ............................................ 72
Viewing ............................ 68, 86
Question marks ..............................72
Results............................................. 70
Quick Read .....................................66
Results............................................. 72
R RNA Quantitation ......................... 78
Read Step ........................................66 S

Reader
Sample Control Wells ................... 81
Connect...................................18
Sample Dilutions ........................... 81
Connecting .............................14
Sample Protocols ........................... 42
Testing ....................................75
Save
Reader..............................................14
Experiment ............................ 58
Reader..............................................18
Protocol .................................. 78
Reader..............................................75
Save ................................................. 62
Reading Procedure
Secure Network ............................. 22
Defining ............................66, 79
Security
Reagent dispensing .............117, 119
File Storage ............................ 20
Reduce database size...................133
User Accounts ....................... 28
Reduction Requirements ..............84
Sensitivity
Repair/compact the database ....133 Lower/Raise........................ 126
Report Builder ..........................86, 87 Sensitivity ..................................... 126
Report Content...............................87 Shared Database ............................ 26
Reporting Engine ...........................87 Show
Reports Reading results...................... 68
Customizing...........................86 Slow response time from Gen5.. 123
Defining ..................................87 Software performance degradation
Meaning of symbols, asterisks ................................................... 123
.............................................72 Spectrum......................................... 70
Rules and Best Practice.........86
142 | Index
ß Fluorescence/Luminescence
Measurements ................ 126
ß-Galactosidase ..............................78
Restore optimal performance
.......................................... 125
S
Troubleshooting .......................... 122
Standard Curve ..............................67 Tutorials.......................................... 42
Standard User.................................29
Statistics...........................................68 U
Symbols and special notations.....72 User Groups ................................... 29
Synergy 2/4 User’s Permissions
Incompatible protocol.........122 User’s Account ...................... 30
Synergy HTTR................................14 Users.......................................... 28, 29
Synergy HTTR w/Injectors ........117
System Administrator V
Changing password..............24
Viewing
Designating ............................18 Results .................................... 68
To Do List ...............................23 Viewing Data
System Administrator ...................22 Customizing views ............... 86
System-generated data reduction 84 Viewing Data ................................. 86
Viewing Preferences ..................... 18
T Viewing Results............................. 68
TAC..................................................56 Vista ................................................ 25
Technical Assistance......................56
Temporary Files .............................24 W
Testing Welcome screen ............................. 44
Reader .....................................75 Well Analysis Data Reduction..... 84
Testing .............................................75 Windows 2000 ............................... 25
Titer Assay ......................................78 Windows Authentication
Toolbar benefits of............................... 34
Buttons and Icons Guide ......52 configuring ............................ 34
Top Probe Vertical Offset ...........126 Gen5 Administrator tasks.... 35
Troubleshooting IT Administrator tasks ......... 36
Communication Errors.......123
requirements.......................... 34
Windows Explorer ........................ 60
| 143
Windows Vista .........................4, 122 Introduction........................... 42

Windows XP ..................................25 Wrong pH..................................... 126
Workspace
144 | Index

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Gen5™ Getting Started

Microplate Data Collection & Analysis

BioTek® Instruments, Inc.

Install Gen5 ..................................................................1

Initial Setup ..................................................................13

Essential Concepts ........................................................57

Basic Tasks ...................................................................65

How to Create a Standard Curve .............................. 67

Setting Up a Protocol ....................................................77

Sample Protocols ..........................................................91

System Administrator’s Password ............................. 130

An “install wizard” guides you through the installation of Gen5; just

Gen5 License Agreement and Warranty Information ........... 2

Gen5 License Agreement and Warranty

BioTek Instruments, Inc. (also referred to as “BioTek”) has developed Gen5

 If your instrument reveals a basecode with a version number

PowerWave XS 1.06 (Any PowerWave XS2 basecode)

Synergy Mx Any Synergy Mx basecode

Clarity NA, but version 4.0.1.10 or higher of Clarity

Installing the Software

 BioTek strongly recommends running a Windows Update to

To install Gen5, follow these steps:

Custom vs. Typical Installation

Gen5 OLE Automation Toolkit

 To learn about security options when using OLE Automation,

Install Other Software Components

Next Steps: Register Gen5 and Initial Setup

Register with BioTek

 As required for installing the software, you must have

 Vista users: If prompted for administrative privileges, engage

Launch Gen5 and Register the Software

 Click Demo to run Gen5 without registering it for the number

4 Click Go to Internet Site.

 If you do not have access to the Internet, contact BioTek (see

Connecting a Reader ..................................................... 14

System > Reader Configuration

 Special note for Clarity users: Settings can be defined only

1 Turn on the reader.

4 Click the Add button to define the Reader Settings.

5 Use the drop-down list to select the Reader Type.

• For first-time setup, Windows may recognize New Hardware and

directions provided with the driver. When the wizard is finished

• PowerWave: This eight-channel monochromator does not need

 Two models of PowerWave XS are listed in Gen5: PowerWave

• ELx800 and ELx808: BioTek configures the onboard software of these

Setting Up the Clarity Luminometer

Clarity users must follow a slightly different sequence of steps to establish

 Install the Clarity PC software before proceeding.

1. Connect the luminometer to the computer (if not already connected)

5. Start Gen5 and log in (if required).

Setting Up Gen5 and Gen5 ELISA

 For Gen5 and Gen5 ELISA Only

Recommended Tasks to Perform

Gen5 System Administrator

For all levels of Gen5 except Gen5 Secure and Gen5CL

How to Change the System Administrator’s Password

 This function is available only to the System Administrator. You

1 Select System> User Setup, and select the Administrator tab.

If your instrument reveals a basecode with a version number

BioTek strongly recommends running a Windows Update to

To learn about security options when using OLE Automation,

As required for installing the software, you must have

Vista users: If prompted for administrative privileges, engage

Click Demo to run Gen5 without registering it for the number

If you do not have access to the Internet, contact BioTek (see

Special note for Clarity users: Settings can be defined only

Two models of PowerWave XS are listed in Gen5: PowerWave

Install the Clarity PC software before proceeding.

For Gen5 and Gen5 ELISA Only

This function is available only to the System Administrator. You

Attention Gen5 Secure users: To ensure 21 CFR Part 11

AutoSave Feature: Gen5 offers this feature to give you

For Gen5 Secure and Gen5CL only

Before modifying a user’s account, make sure he/she is not

Do not forget the Administrator’s password. If you do, you’ll

The QC Trending database, installed with the QC Trending

You may need to change your operating system settings to view

During regular installation, Gen5 Secure installs and enables the

Unlocking a user’s account following an automatic lock resets its

The default settings shipped with Gen5 Secure, and shown in

BioTek has validated this feature on Microsoft Windows Server

To verify that the email server is configured correctly to support