Agilent Oligo Pro II User Manual

Oligo Pro II

User Manual

Notices

CAUTION

WARNING

Document Information

Document No: D0002114 Rev. A EDITION 05/2020

No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.

Agilent Technologies, Inc. 5301 Stevens Creek Blvd. Santa Clara, CA 95051

Trademark acknowledgements

Oligo Pro II is a trademark of Agilent Technologies, Inc.

ProSize software is a registered trademark of Agilent Technologies, Inc.

Windows is a registered trademark of Microsoft Corporation.

Revisions and Updates

This manual is subject to change without notice. This manual may be revised and updated periodically as components and/or maintenance procedures are modified. These updates will be provided to all manual holders on record. Contact Agilent if you have questions regarding availability of updates. Please take care to keep your contact information current with Agilent so that revisions and updates can be provided in a timely manner.

Warranty

The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agreement shall control.

Technology Licenses

The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license.

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252.227-7015 (Technical Data Commercial Items) and DFARS

227.7202-3 (Rights in Commercial Computer Software or Computer Software Documentation).

Safety Notices

A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met.

In This Guide

Agilent has prepared this manual as a technical reference for the Oligo Pro II systems.

This document includes system overviews, analytical methods, maintenance procedures, software operation, troubleshooting guide, and instrument shutdown procedures. Additional information includes literature references, instrument specification and utility requirements, parts and supply lists, product specification sheets, and system warranty information.

This document is intended for use by technical personnel that are proficient with analytical instrumentation operation and upkeep. A certain level of training and expertise is assumed and fundamentals are not addressed herein. Procedures are presented in a step-by-step format using photos and screen captures. If questions remain after reviewing a given topic or procedure, please contact your corresponding Agilent Sales/Service Representative.

1 Oligo Pro II System Overview

This chapter gives an instrument overview.

2 Oligo Pro II Software – File Menu

This chapter describes the Oligo Pro II software in more detail on the commands of the File menu.

3 Oligo Pro II Software – Admin Menu

This chapter describes the Oligo Pro II software in more detail on the commands of the Admin menu.

4 Oligo Pro II Software – Utilities Menu

This chapter describes the Oligo Pro II software in more detail on the commands of the Utilities menu.

5 Oligo Pro II Software – Help Menu

This chapter describes the Oligo Pro II software in more detail on the commands of the Help menu.

Oligo Pro II User Manual 3

6 Oligo Pro II Software – Operation Tab

This chapter describes the Oligo Pro II software in more detail on the Operation tab.

7 Oligo Pro II Software - Run Status Tab

This chapter describes the Oligo Pro software in more detail on the Run Status tab.

8 Oligo Pro II Software – Sample Name Entry

This chapter provides information on how to enter the sample names in the Oligo Pro II software.

9 Oligo Pro II Capillary Array

This chapter explains the essential operational parameters of the capillary array.

10 Appendix

This chapter provides a Quick Start Guide and additional information on part numbers, maintenance procedures, and system settings.

4 Oligo Pro II User Manual

Content

1 Oligo Pro II System Overview 7

About the System 8

Configured Oligo Pro II System Dimensions 9

Oligo Pro II System Connections 10

Oligo Pro II External Cabinet 12

Top Compartment 13

Side Compartment 14

Drawers 16

Oligo Pro II System Loading and Orientation of 96-Well Plates 18

Oligo Pro II Loading Samples 19

2 Oligo Pro II Software – File Menu 20

About this Software 21

System Requirements 22

System Installation 23

Opening the Oligo Pro II Software 24

Main Screen Toolbar 26

File Menu 27

3 Oligo Pro II Software – Admin Menu 30

Admin Menu 31

4 Oligo Pro II Software – Utilities Menu 45

Utilities Menu 46

5 Oligo Pro II Software – Help Menu 56

Help Menu 57

6 Oligo Pro II Software – Operation Tab 59

Operation Tab Overview 60

Oligo Pro II User Manual 5

Content

7 Oligo Pro II Software - Run Status Tab 75

Run Status Tab Overview 76

8 Oligo Pro II Software – Sample Name Entry 83

Sample Name Entry 84

9 Oligo Pro II Capillary Array 90

Capillary Array Parts 91

Removal of the Capillary Array 92

Unpacking a New Capillary Array 101

Capillary Array Installation 107

10 Appendix 115

Permissible Characters 116

Preventative Maintenance Schedule 117

Capillary Array Window Cleaning 118

Oligo Pro II Quick Start Guide 119

Compatible Plates for the Oligo Pro II System 120

6 Oligo Pro II User Manual

1 Oligo Pro II System Overview

About the System 8

Configured Oligo Pro II System Dimensions 9

Oligo Pro II System Connections 10

Oligo Pro II External Cabinet 12

Top Compartment 13

Side Compartment 14

Drawers 16

Oligo Pro II System Loading and Orientation of 96-Well Plates 18

Oligo Pro II Loading Samples 19

This chapter gives an instrument overview.

Oligo Pro II User Manual 7

1 Oligo Pro II System Overview

About the System

The Oligo Pro II system is a multiplexed capillary electrophoresis (CE) instrument for performing automated, high throughput separation and quantification of single-stranded nucleic acids. Separation is achieved by applying an electric field through a narrow bore (75 µm i.d.) fused silica capillary array filled with various conductive gel matrices designed to sieve DNA/RNA molecules of a specific size range. When a high voltage is applied to the capillary array, injected DNA/RNA migrates through the gel matrix as a function of length or size, with smaller sized fragments eluting faster than larger sized fragments.

At a point toward the far end of the capillary array, detection of the separated DNA/RNA is achieved by UV absorption spectroscopy. By monitoring the relative UV absorption as a function of time during the CE separation, digital electropherograms representative of the DNA/RNA content of 12, 24, or 96 samples are collected in a single experimental run.

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1 Oligo Pro II System Overview

38” W (96.52 cm)

26.5” H (67.31) cm)

19” W (48.26 cm) + 24” D (61 cm)

Configured Oligo Pro II System Dimensions

This chapter provides a basic overview of the Oligo Pro II system hardware and operation. Figure 1 shows an external view of a fully configured Oligo Pro II system, which has a compact footprint of 40” on the bench top with a weight of 82 lbs (37 kg).

Figure 1 Configured Oligo Pro II system with computer workstation

Oligo Pro II User Manual 9

1 Oligo Pro II System Overview

NOTE

Oligo Pro II System Connections

The back of the Oligo Pro II instrument contains the communications panel where necessary connections are made to the instrument computer and electrical outlet for operation (Figure 2 and Figure 3).

The use of a double-conversion surge protection or uninterrupted power supply (UPS) device is highly recommended. Contact the corresponding Agilent Sales/Service representative for specific recommended models.

A minimum of three standard electrical wall outlets should be available to connect the instrument, computer and accessories, although a power strip can be used in place of separate wall outlets if needed.

Each connection is labeled on the PC. The various connections between the system and the Oligo Pro II instrument are summarized below:

• From the Oligo Pro II System:

• Two USB cables to PC USB

• Power cord to grounded electrical outlet

• From the PC:

• Two USB connections to the Oligo Pro II system

The order/location of connections is critical and the locations have been identified on the computer.

• Power cord to grounded electrical outlet

• Connection to monitor, keyboard, mouse etc.

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1 Oligo Pro II System Overview

AC power connection

USB connections from instrument

Internet connection

Computer monitor connection

USB out to computer

AC power connection

Fuse mount

Power switch

Oligo Pro II System Connections

Figure 2 Back panel computer connections

Oligo Pro II User Manual 11

Figure 3 Back panel Oligo Pro II instrument connections

1 Oligo Pro II System Overview

Top compartment: Capillary array Optics Power supply

Drawers

Side compartment: Pump Reagent bottles

Oligo Pro II External Cabinet

There are three primary points of access to the inside of the Oligo Pro II system: the top compartment, the side compartment access door, and the drawers (6 total) (Figure 4).

12 Oligo Pro II User Manual

Figure 4 Entry points of the Oligo Pro II System

1 Oligo Pro II System Overview

Capillary array

High voltage supply cable

Capillary outlet connection

Reservoir

Top C om p ar tme nt

Top Compartment

The top compartment provides access to the optical detection platform and a capillary array cartridge. A non-accessible compartment on the back of the instrument contains the high voltage power supply and electronics that are connected to the array cartridge and safety interlock system. The safety interlock system shuts off the high voltage in case this door is opened while the instrument is running.

The capillary array cartridge is a replaceable, modular component of the Oligo Pro II system. You can easily exchange the capillary array cartridge. This process is explained in Chapter 4, “Oligo Pro II Software – Utilities Menu”.

Oligo Pro II User Manual 13

Figure 5 Oligo Pro II main unit top compartment open

1 Oligo Pro II System Overview

6-way distribution valve

Conditioning solution

Waste

Gel 1

Side Compartment

The side compartment allows access to the high pressure pump, syringe, waste bottle, conditioning solution, and gel solution.

The High-Pressure Syringe Pumping system provides automated flushing and filling of the capillary array with conditioning solution and separation gel, providing pressurization of the capillaries up to 280 psi.

Two different solutions are fed to and pumped through the capillary array during routine operation:

• Capillary Conditioning Solution

• Separation Gel (gel 1)

The appropriate solution is selected for pumping by way of a 6-way distribution valve.

The system also contains a waste bottle, which collects solutions pumped via the waste line from the capillary array reservoir during the filling process.

14 Oligo Pro II User Manual

Figure 6 Side door compartment

1 Oligo Pro II System Overview

Side Compartment

The four fluid line connections inside the Oligo Pro II system are:

• Gel line from syringe pump to gel bottle (gel 1 or gel 2)

• Conditioning fluid from syringe pump to conditioning fluid

• Overflow waste line from syringe pump to waste bottle

• F-port line from syringe pump (6-way valve) to F-port

Oligo Pro II User Manual 15

1 Oligo Pro II System Overview

Drawers

The Oligo Pro II system front-panel drawers provide an external interface for loading Buffer, Waste, Rinse, and Sample 96-Well Plates into the system.

• Buffer drawer (top drawer): This location is used for the inlet buffer tray used

during the CE separation.

• Waste drawer (second drawer from top): This location is used for loading the

waste tray.

• Rinse drawer (third drawer from top): This location is used for loading the

rinse tray.

• Sample drawer 1(fourth drawer from top): This location is used for sample

plate number 1.

• Sample drawer 2 (fifth drawer from top): This location is used for sample plate

number 2.

• Sample drawer 3 (sixth drawer from top): This location is used for sample

plate number 3. It is also used for a 96-well plate containing sample storage solution.

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1 Oligo Pro II System Overview

Buffer drawer

Waste drawer

Rinse drawer

Drawer 2: sample

Drawer 3: sample

Drawers

Drawer Status

Status Description

Buffer and Waste Drawers are interlocked When any of the top two drawers are open, the

Rinse, Sample Drawers 1, 2, and 3 are not interlocked Sample trays can be exchanged while the instrument

high-voltage (for electrophoresis) will automatically shut off.

is in operation.

Oligo Pro II User Manual 17

Drawer 1: sample

Figure 7 Instrument drawer positions

1 Oligo Pro II System Overview

NOTE

Well A1 (capillary 1)

Oligo Pro II System Loading and Orientation of 96-Well Plates

The Oligo Pro II system is a multiplexed CE system containing a capillary array, which is designed to interface directly with an entire plate of a standard 96-well plate footprint.

Each capillary of the array corresponds to a specific well for a given row in the 96-well sample plate. For example, the capillary array orientation is indexed such that capillary #1 corresponds to Well A1 and capillary #96 = H12.

Well A1 of the 96-well plate should always be oriented to the back left location of the instrument drawer to ensure that the sample well location is correctly assigned and reported in the software.

18 Oligo Pro II User Manual

Well H12 (capillary 96)

Figure 8 Proper orientation when loading 96-well buffer and sample plates for a 96-capillary system

Each drawer location houses a tray carrier containing alignment pins for ensuring proper alignment of the 96-well plate when placed against the capillary array.

The Oligo Pro II system has been designed to operate using specific dimensions and styles of plates.

Plates with similar dimensions may be used, but capillary damage may occur with the use of poor-quality PCR plates.

For a list of compatible PCR plates please refer to “Compatible Plates for the

Oligo Pro II System” on page 120.

1 Oligo Pro II System Overview

NOTE

Oligo Pro II Loading Samples

The Oligo Pro II system requires a minimum volume of 20 µL/well in the sample plate for proper injection.

When preparing sample plates for repeated use, a volume of 30 µL/well with a 20 µL (one drop) mineral oil overlay is recommended.

Check the wells of the sample plate/s after pipetting to ensure that there are no air bubbles trapped in the bottom of the wells. The presence of trapped air bubbles can lead to injection failures.

Air bubbles can be removed from the plates by introducing a brief centrifugation step prior to placing the plates into the tray carrier.

Oligo Pro II User Manual 19

2 Oligo Pro II Software – File Menu

About this Software 21

System Requirements 22

System Installation 23

Opening the Oligo Pro II Software 24

Main Screen Toolbar 26

File Menu 27

File Manager 27 Oligo Pro II Data Analysis 29 Logout 29 Exit 29

This chapter describes the Oligo Pro II software in more detail on the commands of the File menu.

Oligo Pro II User Manual 20

2 Oligo Pro II Software – File Menu

About this Software

The Oligo Pro II system employs proprietary software for operation and data analysis.

This software is preloaded on the instrument and checked prior to shipment as part of the instrument verification.

Oligo Pro II User Manual 21

2 Oligo Pro II Software – File Menu

System Requirements

The software is run using a Windows 10 PC with the following requirements:

Table 1 Minimum computer requirements

Type Specification

Processor Intel Core i5 - 8500

SVGA Video Minimum Resolution 1024 X 768

Memory 4 Gigabytes (1 x 4 GB) DDR4-2666

Available Hard Disk Space 500 Gigabytes

USB Serial Ports 6 ports (2 instrument, keyboard, mouse)

Network If not using a local database, a network connection to the database

server host is desired.

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2 Oligo Pro II Software – File Menu

System Installation

To install the Oligo Pro II software: 1 Contact your corresponding Agilent Service Representative to request the

software.

2 Navigate to Oligo Pro II Installer > setup.exe, and double-click setup.exe.

Use the appropriate downloaded .exe to perform the installation.

3 Follow the setup instructions provided by the installation wizard. The default

installation directory is C:\Agilent\ Oligo PRO II.

Oligo Pro II User Manual 23

2 Oligo Pro II Software – File Menu

Opening the Oligo Pro II Software

1 To log into the software, select the Oligo Pro II software icon (Figure 9).

Figure 9 Oligo Pro II software icon

There are two levels of users available:

• Administrator: The administrator login has enhanced access to functions

such as allowing the user to edit separation methods.

•User: The user login has restricted access that allows only routine

operation of the instrument.

2 To log into the Oligo Pro II software, type Administrator or User into the User ID

field of the login window (Figure 10).

3 Enter your password.

The first time you log in, the password information is left blank.

Figure 10 Login menu

24 Oligo Pro II User Manual

2 Oligo Pro II Software – File Menu

Opening the Oligo Pro II Software

4 Click OK.

The main screen opens.

Figure 11 Oligo Pro II software main screen

More information about the User and Administrator functions within the software are discussed in a later chapter.

A password can be set for the system during the Agilent training and installation period at your facility or by using the Change Password command of the Admin menu described in section “Change Password” on page 38.

The first time you log into the Oligo Pro II software, a database is displayed indicating where the application is connected.

The database connection can be changed before logging in by clicking Browse next to the database field.

The login information is used in event and error logging to aid in controlling access to the system, tracking usage and monitoring changes to the system.

Every time you log into the Oligo Pro II software, you begin on the main screen (Figure 11).

Oligo Pro II User Manual 25

2 Oligo Pro II Software – File Menu

Main Screen Toolbar

The main screen toolbar is located at the top of the Oligo Pro II software main screen as seen in Figure 11.

26 Oligo Pro II User Manual

2 Oligo Pro II Software – File Menu

File Menu

Figure 12 File menu functions

File Manager

The File manager command allows electropherogram data to be examined within the Oligo Pro II software environment.

Files are normally analyzed using the Oligo Pro II Data Analysis software, which is covered in the Oligo Pro II Data Analysis Software User Manual.

The file manager also enables you to correct the capillary alignment for an individual data file.

Select the File manager command to open a file browser and to navigate to a data file. Once a file is selected, the file manager window opens (Figure 13).

Oligo Pro II User Manual 27

2 Oligo Pro II Software – File Menu

File Manager

Figure 13 File manager window

Tabl e 2 lists the functions of the file manager screen.

Table 2 File manager – file functions

Field Description

Open Opens a file browser to navigate to desired data file.

Cap. Alignment Allows you to view and manipulate the capillary alignment for the data file opened

only. Capillary alignment from a file is discussed in section “Capillary Alignment” on page 47.

Print Allows you to print the twelve electropherograms to a page.

Exit Closes the File manager window.

Tabl e 3 lists the Current, Method summary, and Sample info toolbar functions.

Table 3 File manager toolbar options

Field Description

Current Displays the current of the separation during the analysis.

Method summary Shows a summary of the method that was used for the separation.

Sample Info Shows the sample names input for the separation file.

28 Oligo Pro II User Manual

2 Oligo Pro II Software – File Menu

Oligo Pro II Data Analysis

Once the data file is opened in the file manager, the data can be viewed in groups of 12 (by row) when the Group tab is selected. A page selection is located at the bottom of the screen allowing for navigation of all rows in a plate (assuming 96-capillary array data is chosen).

To view a single electropherogram at a time, either left-click twice on the desired well or select the Single tab. A page and well selection is located at the bottom of the screen allowing for navigation of all rows and wells in the plate.

Electropherogram data can be panned, zoomed in, or zoomed out of by right-clicking on the chart and selecting the function of interest.

Oligo Pro II Data Analysis

Selecting this command will open the Oligo Pro II data analysis software.

Logout

The Logout command allows you to log out of the Oligo Pro II software and to log in as a different user.

After logout, the login menu opens (Figure 10).

Exit

The Exit command closes the Oligo Pro II software. Alternatively, you can exit the program by selecting the red X on the top right corner of the main screen.

Oligo Pro II User Manual 29

3 Oligo Pro II Software – Admin Menu

Admin Menu 31

Configuration 32 Change Password 38 User Maintenance 39 Archive and Purge Database 40 Event Report 41 Error Report 43

This chapter describes the Oligo Pro II software in more detail on the commands of the Admin menu.

Oligo Pro II User Manual 30

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