Thermo Fisher Scientific 7900HT Fast Real-Time PCR Systems Owner's Manual

Applied Biosystems 7900HT Fast

Real-Time PCR System

Maintenance and

Troubleshooting Guide

Performing

Calibration and

Verification Runs

Maintaining the

7900HT

Instrument

Hardware

Maintaining the

Computer and

Software

Maintaining the

Automation

Accessory

Troubleshooting

Chemistry and

Assay Runs

Flags and Filtering

© Copyright 2007,

2010 Applied Biosystems. All rights reserved.

For Research Use Only.

Not for use in diagnostic procedures.

Notice to Purchaser

The Applied Biosystems 7900HT Fast Real-Time PCR System is a real-time thermal cycler covered by US p atents and corresponding claims in their non-US counterparts, owned by Appl ied Biosystems . No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5’ nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of

Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

TRADEMARKS:

ABI PRISM, Applied Biosystems, MicroAmp, and VIC are registered trademarks and AB (Design), Applera, FAM, JOE, NED, ROX, TAMRA, and TET are trademarks of Appli ed Biosystems or its subsidiaries in the U.S. and/or certain other countries.

GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems, Inc.

SYBR is a registered trademark of Molecular Probes, Inc.

Zymark is a registered trademark of Zymark Corporation.

Windows is a registered trademark of Microsoft Corporation.

All other trademarks are the sole property of their respective owners.

Part Number 4365542 Rev. C

06/2010

Contents

Preface v

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Chapter 1

Performing Calibration and Verification Runs 1

Recommended Run Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Performing a Background Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Performing a Pure Dye Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Performing an Instrument Verification Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Chapter 2

Maintaining the

7900HT Instrument Hardware 51

Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Removing and Installing a Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Replacing the Plate Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Chapter 3

Maintaining the Computer and Software 71


Archiving and Backing Up SDS Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Defragmenting the Hard Drive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Managing Local User Accounts in the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . 78

Updating the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Updating the Operating System Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Troubleshooting Software and Computer Problems . . . . . . . . . . . . . . . . . . . . . . . . . 84

Chapter 4

Maintaining the Automation Accessory 87


Automation Accessory Components and Stack Positions . . . . . . . . . . . . . . . . . . . . 89

Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Adjusting the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

7900HT Fast System Maintenance and Troubleshooting Guide

iii

Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Troubleshooting the Automation Accessory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Chapter 5

Troubleshooting Chemistry and Assay Runs 119

Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Standard Curve (AQ) and Comparative CT (RQ) Quantitative Assay Runs . . . . . . 124

Allelic Discrimination and Plus/Minus End-Point Assay Runs . . . . . . . . . . . . . . . . 126

Chapter 6

Flags and Filtering 127

Using Flags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Flags By Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Description of Flags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Appendix A

Adding Custom Dyes to the Pure Dye Set 141

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Creating a Dilution Series Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Creating a Plate Document for the Dilution Series Plate . . . . . . . . . . . . . . . . . . . . 144

Running and Analyzing the Dilution Series Plate . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Creating a Pure Dye Plate with the Custom Dye(s) . . . . . . . . . . . . . . . . . . . . . . . . . 148

Adding the New Custom Dye(s) to the SDS Software . . . . . . . . . . . . . . . . . . . . . . 149

Creating a Plate Document Template with the Custom Dye(s) . . . . . . . . . . . . . . . . 150

Appendix B

Instrument Connections 153

Appendix C

Parts and Consumables 157

Interchangeable Sample Block Modules and Accessories . . . . . . . . . . . . . . . . . . . 157

Consumables and Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Instrument Maintenance and Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Index 163

iv


Preface

How to Use This Guide

Purpose of This

Guide

This guide provides recommended schedules and procedures for maintaining the

Applied Biosystems 7900HT Fast Real-Time PCR System (7900HT Fast System). This guide also includes troubleshooting information for the maintenance procedures.

Audience

This guide is intended for:

• Personnel who maintain the 7900HT Fast System hardware

• System Administrators who maintain the 7900HT Fast System software

• Personnel running experiments

Assumptions

For hardware maintenance, this guide assumes that you have:

• Familiarity with the Microsoft Windows

®

XP operating system.

• Knowledge of general techniques for handling DNA samples and preparing them for PCR.

For software maintenance, this guide assumes that you have:

• Familiarity with the Microsoft Windows

®

XP operating system.

• A general understanding of hard drives and data storage, file transfers, and copying and pasting.

• Networking experience, if you want to integrate the 7900HT Fast System into your existing laboratory data flow

Text Conventions

This guide uses the following conventions:

• Bold indicates user action. For example:

Type 0, then press Enter for each of the remaining fields.

• Italic text indicates new or important words and is also used for emphasis. For example:

Before analyzing, always prepare fresh matrix.

• A right arrow bracket (>) separates successive commands you select from a dropdown or shortcut menu. For example:

Select File > Open.

User Attention

Words

The following user attention words appear in Applied Biosystems user documentation.

Each word implies a particular level of observation or action as described below:

Note

– Provides information that may be of interest or help but is not critical to the use of the product.


v

Preface

How to Obtain More Information

IMPORTANT!

– Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical.

Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices.

Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury.

Safety

Follow specific safety practices when using this instrument. For safety guidelines, refer to the “Safety and EMC Compliance” section in the Applied Biosystems 7900HT Fast

Real-Time PCR System Site Preparation Guide (PN 4351923).

For any chemical manufactured or distributed by Applied Biosystems, you can obtain the MSDS from Applied Biosystems. This service is available free 24 hours a day. To obtain MSDSs:

1. Go to

www.appliedbiosystems.com

, click Support, then click MSDS Search.

2. In the Keyword Search field, enter the chemical name, product name, MSDS part number, or other information that appears in the MSDS of interest. Select the language of your choice, then click Search.

3. Find the document of interest, right-click the document title, then select any of the following:

• Open – To view the document

• Print Target – To print the document

• Save Target As – To download a PDF version of the document to a destination that you choose

For the MSDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer.

How to Obtain More Information

Related

Documentation

For more information about using the 7900HT Fast System, refer to:

• Sequence Detection Systems Software version 2.3 Online Help (SDS Online Help)

• Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation Guide

(PN 4351923)

• Applied Biosystems 7900HT Fast Real-Time PCR System Allelic Discrimination

Getting Started Guide (PN 4364015)

• Applied Biosystems 7900HT Fast Real-Time PCR System Absolute Quantitation

Using Standard Curve Getting Started Guide (PN 4364014)

• Applied Biosystems 7900HT Fast Real-Time PCR System Plus-Minus Getting

Started Guide (PN 4364017)

• Applied Biosystems 7900HT Fast Real-Time PCR System Relative Quantitation

Using Comparative C

T

Getting Started Guide (PN 4364016)

• Real-Time PCR Systems Chemistry Guide (PN 4348358)

vi


Preface

How to Obtain Support

• SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time PCR

System Administrators Guide (PN 4351669)

• TaqMan

®

Low Density Array Getting Started Guide (PN 4319399)

Send Us Your

Comments

Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to:

[email protected]

How to Obtain Support

To contact Applied Biosystems Technical Support from North America by telephone, call 1.800.762.4001.

For the latest services and support information for all locations, go to

http://www.appliedbiosystems.com

, then click the link for Support.

At the Support page, you can:

• Obtain worldwide telephone and fax numbers to contact Applied Biosystems

Technical Support and Sales facilities

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support

• Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents

• Download PDF documents

• Obtain information about customer training

• Download software updates and patches


vii

Preface

How to Obtain Support

viii


Chapter 1

Performing Calibration and

Verification Runs

1

Performing

Calibration and


Maintaining the 7900HT

Instrument

Hardware

Maintaining the

Computer and

Software

Recommended

Run Schedule

See page 2

Performing a

Background Calibration

See page 3

Maintaining the

Automation

Accessory

Performing a

Pure Dye Calibration

See page 17

Troubleshooting

Chemistry and

Assay Runs


Notes


Performing an

Instrument Verification Run

See page 32

1

Chapter 1 Performing Calibration and Verification Runs

Recommended Run Schedule

Recommended Run Schedule

To ensure optimal performance of your 7900HT Fast System, perform the calibration and verification runs as indicated in the table below.

Schedule

Semi-annually

January

Su M T W Th F S

July

Su M T W Th F S

6 Months

Run Type

Perform a background calibration (see page 3

)

IMPORTANT!

You must also perform a background calibration before performing a pure dye calibration and after installing a new, uncalibrated sample block.

Perform a pure dye calibration (see

page 17

)

IMPORTANT!

You must also perform a pure dye calibration before performing an instrument verification run and after installing a new, uncalibrated sample block.

Perform an instrument verification run (see page 32

)

Notes

2



Performing a Background Calibration

Performing a Background Calibration

Overview

When to Perform

Applied Biosystems recommends performing a background calibration:

• Every 6 months, or as often as needed depending on instrument use

• Before performing a pure dye calibration (see

page 17 )

• After installing a new, uncalibrated sample block (see

page 53 )

You can also perform background calibrations to detect and troubleshoot sample block contamination.

About the

Background

Calibration

A background calibration measures the level of ambient fluorescence in the 7900HT instrument. During the run, the 7900HT instrument performs a continuous scan of a background plate (which contains PCR buffer or deionized water) for 2 minutes at

60 °C. Afterwards, the SDS software averages the spectrum recorded during the run and extracts the resulting spectral component to a calibration file. The SDS software uses the calibration file during subsequent runs to remove the background signal from the run data.

About the

Background

Component

Fluorescence collected by the 7900HT Fast System includes a fluorescent signal inherent to the system, commonly referred to as background fluorescence. This background component is a composite signal found in all spectral data that consists of fluorescence from several sources, including: the background electronic signal, the sample block, the water inside the consumable, and the consumable itself. Because the background fluorescence can interfere with the precision of the data collected by the

7900HT System, the instrument is engineered to minimize the background signal; additionally, the SDS software algorithmically eliminates the background signal from each fluorescent sample to maximize the instrument’s sensitivity.

1

Notes


3



Before You Begin

Time Required

30 minutes

Materials

Required

You need the materials listed below to perform this procedure.

For 384- or 96-Well Sample Blocks

Powder-free gloves and safety goggles

Background plate from one of the following calibration kits:

• Sequence Detection Systems 384-Well

Spectral Calibration Kit (PN 4323977)

• ABI P

RISM

®

7900HT Sequence Detection

Systems 96-Well Spectral Calibration Kit

(PN 4328639)

• 7900HT System Fast 96-Well Spectral

Calibration Kit (PN 4351653)

Centrifuge, with plate adapter

For the TaqMan Low Density Array Block


7900HT System Fast 96-Well

Spectral Calibration Kit

Part No: 4351653

Lot No: 0409003

Store at -15°C to -25°C

Caution!

May cause eye, skin, and respiratory track irritation.

Made in USA

TaqMan

®

Low Density Array Spectral

Calibration Kit (PN 4362745). Use:

• The tube of background solution

• One empty TLDA

Spectral Calibrati

Part No: 0000001

Lot No:

Made in

USA

1000002

7900HT

TaqMan

®

Density Empty

Low

Array

0000001

Part No:

Lot No: 1000002

For Research Use Only

Not for use in

.

diagnostic procedures

Notes

4




Vortex

Micropipettors and pipet tips:

• Rainin F-100 micropipette (100-

µL)

• Rainin Fine Point pipet tips (100-

µL)

Centrifuge (For more information, see the

TaqMan

®

Low Density Array Getting Started

Guide.)

GR2170

TaqMan

®

Low Density Array Sealer (For more information, see the TaqMan

®

Low Density

Array Getting Started Guide.)

Workflow

To perform the background calibration:

1.

Obtain the tools and materials required (see pages 4

and

5

).

2.

Prepare the background plate (see page 6 ) or background TLDA (see

page 9

).

3.

Create a plate document (see page 10 ).

4.

Run the background plate or background TLDA (see page 12 ).

5.

Analyze the background data (see page 14 ).

Troubleshooting

If you experience problems with the background calibration, see

“Troubleshooting

Background Calibrations” on page 15

.

1

Notes


5



Preparing the Background Plate

IMPORTANT!

Wear powder-free gloves when you handle the background plate.

1.

Retrieve the calibration kit from the freezer and remove the packaged background plate from within it.

2.

Return the calibration kit to the freezer.

3.

Allow the background plate to warm to room temperature (approximately 5 min).

4.

Remove the background plate from its packaging.

IMPORTANT!

Do not discard the packaging for the background plate. The background plate can be used repeatedly if it is stored in its original packaging sleeve.

Ambient >00:05:00

5.

Briefly centrifuge the background plate in a centrifuge with plate adapter.

<1500

×g

00:00:05

Notes

6




6.

Verify that the liquid in each well of the background plate is positioned at the bottom of the well. If not, centrifuge the plate again at a higher rpm and for a longer period of time.

IMPORTANT!

Do not allow the bottom of the background plate to become dirty. Fluids and other contaminants that adhere to the bottom of the plate can contaminate the sample block and cause an abnormally high background signal.

Correct Incorrect

Sample is positioned at bottom of well.

The plate has not been centrifuged with enough force, or has not been centrifuged for enough time.

7.

Proceed to

“Creating a Background Plate

Document” on page 10

.

1

Notes


7



Creating a Background Plate

If a background plate from a calibration kit is not available, you can create one to perform the background calibration by following the procedure below. Whenever possible, Applied Biosystems recommends the use of the background plate included with the calibration kit because it contains a PCR buffer that more accurately models the reagents used for PCR, and therefore produces better calibration data.

Materials Required

Pipettor, 100 to

200-

µL (with pipet tips)

Deionized water

Safety glasses

Note:

Reaction plate options are:

• 384-Well Clear Optical Reaction Plate (standard

384-well reaction plate)

• MicroAmp

®

96-Well Optical Reaction Plate

(standard 96-well reaction plate)

• Optical 96-Well Fast Thermal Cycling Plate

(Fast 96-well reaction plate)

Reaction plate

(see note at right)

Optical adhesive cover or Optical Caps

(PN 4323032)

To create the background plate:

Powder-free gloves

IMPORTANT!

Wear powder-free gloves while creating the background plate.

1. Remove a reaction plate from its box and place it on a clean, dry surface.

2. Aliquot deionized water to each well of the reaction plate, as follows:

– 20

µL per well for a standard 384-well or Fast 96-well reaction plate

– 50

µL per well for a standard 96-well reaction plate

Pipet deionized water to each well

3. Seal the plate:

– For standard 384-well or Fast 96-well reaction plates, use only an optical adhesive cover.

– For standard 96-well reaction plates, use an optical adhesive cover or optical flat caps.

Seal the plate

4. Continue with step 5 on page 6 .

Notes

8


Preparing the Background TLDA



1.

Retrieve the calibration kit from the freezer and remove:

• The tube of background solution

• One empty TLDA

2.


Calibration reagents box

Empty TLDA

1

Background solution

3.

Allow the background solution to thaw at room temperature.

4.

When the solution has thawed, vortex the tube.

5.

Load the background solution into the empty

TLDA, loading 100

µL of solution per fill reservoir.

Note:

TaqMan

Guide.

For detailed loading procedures see the

®


6.

Centrifuge and seal the background TLDA.

Note:

For centrifuging and sealing procedures see the TaqMan

Started Guide.

®

Low Density Array Getting

7.

Proceed to

“Creating a Background Plate


.

Ambient >00:05:00

Notes


9



Creating a Background Plate Document

1.

Double-click on the desktop (or select Start

> All Programs > Applied Biosystems > SDS

2.3 > SDS 2.3) to start the SDS software.

2.

If the login option is enabled, the Login dialog box appears. Enter your User Name and

Password, then click OK.

Note:

If the login option is not enabled, no

Login dialog box appears. Skip to step 3 below.

3.

Click

4.

Complete the New Document dialog box:

a.

Assay – Select Background.

b.

Container – Select the appropriate format.

c.

Template – Select the appropriate template, as described in the table below:

For Container...

384 Wells Clear Plate

96 Wells Clear Plate (for a Standard 96-Well

Sample Block)

96 Wells Clear Plate (for a Fast 96-Well Sample

Block)

384 Wells TaqMan Low

Density Array

Select Template...

Blank Template

Blank Template

Fast 96 Well Background Plate.sdt

TaqMan Low Density Array

Background Plate.sdt

d.

If the background plate or background

TLDA is labeled with a barcode, click the

Barcode field, then type in or scan the barcode number.

e.

Click . The software creates and opens a plate document with the attributes for a background calibration.

IMPORTANT!

Do not modify the background plate document. The method for a background calibration is coded into the SDS software and consists of a single

Notes

10

4d

4e

4a

4b

4c




hold at 60 °C for 2 min. Because the plate contains only PCR buffer or deionized water, the plate document does not require sample or detector labels.

5.

Save the plate document:

a.

Click (or select File Save) to open the

Save As dialog box.

b.

If the Save in field does not display SDS

Documents, navigate to Applied

Biosystems SDS Documents.

c.

In the File name field, type in an appropriate file name, as described in the table below.




Sample Block)


Block)


Density Array

Type...

Background_<date in MMDDYY

format>

For example, the file name for a plate run on May 31, 2005, would be:

Background_053105.

Background_<date in MMDDYY

format>

For example, the file name for a plat run on May 31, 2005, would be:

Background_053105.

FastBackground_Plate_<date in

MMDDYY format>

For example, the file name for a Fast plate run on May 31, 2005, would be:

FastBackground_053105.

Background<date in MMDDYY

format>_TLDA

For example, the file name for a background TLDA run on

May 31, 2005, would be:

Background_053105_TLDA.

d.

Select SDS 7900HT Document (*.sds) from the Files of type drop-down list.

e.

Click document.

. The software saves the plate

6.

Continue with “Running the Background Plate or

Background TLDA” on page 12

.

5b . This should be

SDS Documents

5c

5e

5d

1

Notes


11



Running the Background Plate or Background TLDA

1.

In the background plate document, select the

Instrument Real-Time tabs.

2.

Click . the OUT position.

1

3.

Place the background plate or background TLDA into the instrument tray as shown.

IMPORTANT!

The background plate or TLDA must be oriented so that the A1 position matches the A1 label on the instrument tray.

2

Standard 384-well reaction plate

Well A1

Barcode

4.

Click . the IN position and performs the background calibration.

Note:

Before starting the run, the instrument may pause (up to 15 min) to bring the heated cover to the appropriate temperature.

5.

When the background calibration is complete and the Run Complete dialog box appears, click to close the dialog box.

Notes

12




6.

Click , then remove the background plate or background TLDA from the instrument tray.

PHYSICAL INJURY

HAZARD. During instrument operation, the sample block can be heated as high as 100 °C.

Before removing the background plate or background TLDA, be sure to wait until the sample block reaches room temperature.

7.

Place the background plate or background TLDA back inside its packaging, then return it to the calibration kit in the freezer.

IMPORTANT!

Do not discard the background plate or background TLDA. If you store it in its original packaging, you can use the background plate or background TLDA for up to 6 months after you open it.

8.

Continue with “Analyzing the Background Data” on page 14

.

-20 to -25

°C

1

Notes


13



Analyzing the Background Data

When you analyze the background data, the SDS software extracts the calibration values from the background plate document, then provides pass/fail information. After extraction, the SDS software stores the data as part of the calibration file located in the

Calibration subdirectory of the SDS directory.

To analyze the background data:

1.

In the Plate Grid of the background plate document, click the box above the A label to select all wells.

Click here

2.

Select Analysis Extract Background.

The software attempts to extract the background signal and displays the success of the extraction in a dialog box.

3.

If the software displays:

Calibration Update Complete – The analysis is successful. The raw spectra read from the background plate conform to acceptable limits.

Click , then go to step 4 .

Error – The run is unsuccessful. The software stopped the extraction because one or more raw spectra

exceed 4000 FSU.

Click , then troubleshoot the failed run as explained in

“Troubleshooting Background

Calibrations” on page 15

.

4.

Click background plate document.

5.

Select File Close to close the background plate document.

Notes

14




Troubleshooting Background Calibrations

Troubleshooting

Table

Table 1-1 Troubleshooting background calibrations

Observation

Software will not extract background data

Possible Cause

During setup, the wrong plate type was assigned to the plate document

Recommended Action

Run a new background plate document with the proper plate type setting.

Background is too high

(>4000 FSU)

Background is too high

(>4000 FSU

‡

)

Sample block contamination

Background plate contamination

See “Isolating Sample Block

Contamination” below.

1. Construct and run a new background plate.

2. See “Isolating

Sample Block

Contamination” below.

‡Fluorescent standard units – The measure of amplitude displayed along the Y-axis of the

Background Plot.

Isolating

Sample Block

Contamination

Signals exceeding 4000 FSU are considered outside the limit of normal background fluorescence and indicates that the either the background plate or the sample block may be contaminated.

1. Open the plate document for the background calibration.

2. In the toolbar, click (Hide/Show System Raw Data Pane).

3. Select all wells in the plate document.

The SDS software highlights the selected wells and displays the raw spectral data.

4. Inspect the raw background data for an aberrant spectral peak or peaks.

Wells producing raw spectra that exceed 4000 FSU are considered irregular and could be contaminated. The following figure illustrates the raw data produced by a run on a sample block module containing a contaminated well.

1

Possible contamination

5.

Drag the vertical bar in the temperature plot to inspect all the data points. The location(s) of the contaminated well(s) is displayed in tooltip when you move the mouse over the curves in the graph.

Notes


15



6. Decontaminate the sample block as explained on

page 65

.

7. Run a background plate to confirm that the contaminants have been removed.

If the contamination is present after running the background plate for a second time, the background plate is likely to be the source of contamination.

Notes

16



Performing a Pure Dye Calibration

Performing a Pure Dye Calibration

Overview

When to Perform

Applied Biosystems recommends performing a pure dye calibration:

• Every 6 months, depending on instrument use

IMPORTANT!

The age and use of instrument components can affect pure spectra readings. Update the pure spectra data files once or twice annually, depending on the frequency of instrument use.

• Before performing an instrument verification run (see page 32 )

• After installing a new, uncalibrated sample block (see

page 53 )

Purpose of the

Pure Dye

Calibrations

Before operating the instrument, you must generate spectral data by performing a pure dye calibration. During the pure dye calibration, the SDS software collects spectral data from a set of pure dye standards during a 2-minute hold at 60 °C. The SDS software stores the spectral data in the pure spectra run file, a calibration file located in the SDS directory. The SDS software uses the spectral data from the set of pure dye standards to distinguish the individual contribution of each dye in the collective fluorescence gathered by the instrument during a run.

After each run, the SDS software receives run data in the form of a raw spectra signal for each reading. To make sense of the raw data, the software must determine the contribution of each fluorescent dye used in the sample by comparing the raw spectra to the spectral data contained in the pure spectra run file.

1 2 3 4 5 6 7

1

2

3

4

5

6

7

Dye

FAM

™

dye

SYBR

®

Green dye

TET

™

dye

VIC

®

dye

JOE

™

dye

NED

™

dye

TAMRA

™

dye

ROX

™

dye

Peak (nm)

~520

~520

~540

~550

~550

~570

~580

~610

Note:

When a plate document is saved after analysis, the SDS software stores the pure spectra information with the collected fluorescent data for that experiment.

Notes

1


17



Components of the Pure Dye

Standards

The 7900HT Fast System is calibrated with several dyes, which are included in the calibration kits.

• The Sequence Detection Systems 384-Well Spectral Calibration Kit is provided for the standard 384-well sample block. This kit includes one 384-well pure dye plate, preloaded with the following dyes: FAM

SYBR

®

Green dye, TAMRA

™

™

dye, JOE

™

dye, NED

dye, TET

™

dye, and VIC

®

dye.

™

dye, ROX

™

dye,

• The ABI P

RISM

®

7900HT Sequence Detection Systems 96-Well Spectral

Calibration Kit is provided for the standard 96-well sample block. This kit includes two 96-well pure dye plates. Plate 1 is preloaded with the following dyes:

FAM

™ dye, JOE

™

dye, NED following dyes: SYBR

®

™

dye, and ROX

™

Green dye, TAMRA

™

dye. Plate 2 is preloaded with the

dye, TET

™

dye, and VIC

®

dye.

• The 7900HT System Fast 96-Well Spectral Calibration Kit is provided for the Fast

96-well sample block. This kit includes two 96-well pure dye plates. Plate 1 is preloaded with the following dyes: FAM

™ dye, JOE

™

dye, NED

™

dye, and ROX

™ dye. Plate 2 is preloaded with the following dyes: SYBR

®

Green dye, TAMRA

™ dye, TET

™

dye, and VIC

®

dye.

• The TaqMan

TaqMan

®

Low Density Array block. The kit includes three tubes of dye: FAM dye, ROX

™

®

Low Density Array Spectral Calibration Kit is provided for the dye, and VIC

®

™ dye and three empty TLDAs. To perform a pure dye calibration, you load each dye into an empty TLDA.

Custom Pure

Dyes

The 7900HT Fast System supports the detection of custom pure dyes (dyes other than those provided by Applied Biosystems). To add custom pure dyes to the pure dye set for your instrument, see

Appendix A

on

page 141

.

Notes

18





Time Required

30 minutes

Materials

Required




Pure dye plate(s) from one of the following calibration kits:

• Sequence Detection Systems 384-Well


• ABI P

RISM

®

7900HT Sequence Detection

Systems 96-Well Spectral Calibration Kit

(PN 4328639)

• 7900HT System Fast 96-Well Spectral

Calibration Kit (PN 4351653)

Note:

Both the standard 96-well and Fast

96-well calibration kits contain two pure dye plates.




7900HT System Fast 96-Well

Spectral Calibration Kit

Part No: 4351653

Lot No: 0409003


Caution!

May cause eye, skin, and respiratory track irritation.

Made in USA

TaqMan

®

Low Density Array Spectral

Calibration Kit (PN 4362745). Use:

• The three dye tubes: FAM

™

dye,

ROX

™ dye, and VIC

® dye

• Three empty TLDAs

Spectral Calibrati

Part No: 0000001

Lot No:

Made in

USA

1000002

7900HT

TaqMan

®

Density Empty

Low

Array

0000001

Part No:

Lot No: 1000002


Not for use in

.

diagnostic procedures

1

Notes


19



Vortex



µL)


µL)


TaqMan

®


Guide.)

TaqMan

®


®

Low Density


GR2170

Notes

20

Workflow

To perform the pure dye calibration:

1.

Perform a background calibration first (see page 3 )

IMPORTANT!

calibration.

You must perform a background calibration before every pure dye

2.


and

20 ).

3.

Prepare the pure dye plate(s) (see

page 21 ) or pure dye TLDAs (see

page 23

).

4.


5.

Run the pure dye plate(s) or pure dye TLDAs (see

page 27

).

6.

Analyze the pure dye data (see

page 29

).

Troubleshooting

If you experience problems with the pure dye calibration, see “Troubleshooting Pure

Dye Calibrations” on page 31

.




Preparing the Pure Dye Plate(s)

IMPORTANT!

You must perform a background calibration before every pure dye calibration.

IMPORTANT!

Wear powder-free gloves when you handle the pure dye plates.

1.

Retrieve the calibration kit from the freezer, then remove the packaged pure dye plate(s).

Note:

The standard 384-well calibration kit contains one pure dye plate. Both the standard

96-well and Fast 96-well calibration kits contain two pure dye plates.

2.


3.

Allow the pure dye plate(s) to warm to room temperature (approximately 5 min).

4.

Remove a pure dye plate from its packaging.

IMPORTANT!

Do not remove a pure dye plate from its packaging until you are ready to run it.

The fluorescent dye contained in the wells of each pure dye plate is photosensitive. Prolonged exposure to light can diminish the fluorescent signal strength of the plate.

IMPORTANT!

Do not discard the packaging for the pure dye plate. The pure dye plate can be used repeatedly if it is stored in its original packaging sleeve.

5.

Briefly centrifuge the pure dye plate in a centrifuge with plate adapter.

Ambient >00:05:00

<1500

×g

00:00:05

Notes


21

1



6.

Verify that the pure dye standard in each well of the pure dye plate is positioned at the bottom of the well. If not, centrifuge the plate again at a higher rpm and for a longer period of time.




7.

Proceed to

“Creating a Pure Dye Plate


.

Notes

22




Preparing the Pure Dye TLDAs

IMPORTANT!

You must perform a background calibration before every pure dye calibration.

IMPORTANT!

Wear powder-free gloves when performing this procedure.

FAM

™

, ROX

™

Spectral Calibration Bulk

, and VIC

®

Dyes are combustible liquids and vapors. Exposure causes eye, skin, and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

1.

Retrieve the calibration kit from the freezer and remove:

• The three dye tubes: FAM dye, ROX dye, and VIC dye

• Three empty TLDAs

2.


Calibration reagents box

Empty TLDAs

1

Dye tubes

3.

Allow the dyes to thaw at room temperature.

4.

When the dyes have thawed, vortex the tubes.

5.

Load the FAM dye into one of the empty

TLDAs, loading 100

µL of dye per fill reservoir.

Note:

TaqMan

Guide.


®


Notes


Ambient >00:05:00

23



6.

Repeat step 5 for the ROX and VIC dyes, using the two remaining empty TLDAs.

7.

Centrifuge and seal the pure dye TLDAs.

Note:


Started Guide.

®


8.

Proceed to

“Creating a Pure Dye Plate


.

Creating a Pure Dye Plate Document

1.




2.



Note:



3.

Click

4.


a.

Assay – Select Pure Spectra.

b.


c.

Template – Select the appropriate template, as described in the table below.




Sample Block)


384 Well Pure Dyes Plate.sdt

• 96 Well Pure Dyes Plate 1.sdt to run Plate 1 (containing FAM, JOE,

NED, and ROX dyes)

• 96 Well Pure Dyes Plate 2.sdt to run Plate 2 (containing SYBR

Green, TAMRA, TET, and VIC dyes)

Notes

24


4d

4e

4a

4b

4c





Block)


Density Array


•

Fast 96 Well Pure Dyes Plate 1.sdt

to run Plate 1 (containing FAM,

JOE, NED, and ROX dyes)

•

Fast 96 Well Pure Dyes Plate 2.sdt

to run Plate 2 (containing SYBR

Green, TAMRA, TET, and VIC dyes)

•


FAM Pure Dye.sdt to run the pure dye TLDA containing FAM dye

•


ROX Pure Dye.sdt to run the pure dye TLDA containing ROX dye

•


VIC Pure Dye.sdt to run the pure dye TLDA containing VIC dye

d.

If the pure dye plate or pure dye TLDA is labeled with a barcode, click the Barcode field, then type in or scan the barcode number.

e.

Click . The software creates and opens a plate document with the attributes for a pure dye calibration.

IMPORTANT!

Do not modify the pure dye plate document.

5.


a.


Save As dialog box.

b.




c.





Sample Block)

Type...

PureDye_<date in MMDDYY format>


PureDye_053105.

PureDye_Plate<plate #>_<date in

MMDDYY format>

For example, the file name for a Plate 1 run on May 31, 2005, would be:

PureDye_Plate1_053105.

Notes


5b . This should be

SDS Documents

5c

5e

5d

25

1





Block)


Density Array

Type...

FastPureDye_Plate<plate #>_<date in

MMDDYY format>

For example, the file name for a Fast

Plate 1 run on May 31, 2005, would be:

FastPureDye_Plate1_053105.

<Dye><date in MMDDYY

format>_TLDA

For example, the file name for a

FAM dye TLDA run on May 31, 2005, would be: FAM_053105_TLDA.

d.


e.

Click document.


6.

Continue with

“Running a Pure Dye Plate or

Pure Dye TLDA” on page 27

.

Notes

26




Running a Pure Dye Plate or Pure Dye TLDA

1.

In the pure dye plate document, select the


2.


1

3.

Place the pure dye plate or pure dye TLDA into the instrument tray as shown.

IMPORTANT!

The pure dye plate or TLDA must be oriented so that the A1 position matches the

A1 label on the instrument tray.

2


Well A1

1

Barcode

4.

Click . the IN position and performs the pure dye calibration.

Note:


5.

When the pure dye calibration is complete and the Run Complete dialog box appears, click to close the dialog box.

Notes


27



6.

Click , then remove the pure dye plate or pure dye TLDA from the instrument tray.

PHYSICAL INJURY


Before removing the background plate or background TLDA, be sure to wait until the sample block reaches room temperature.

7.

Place the pure dye plate or each pure dye TLDA inside its packaging, then return it to the calibration kit in the freezer.

IMPORTANT!

Do not discard the pure dye plate or pure dye TLDAs. If you store it in its original packaging, you can use the pure dye plate or each pure dye TLDA for up to 6 months after you open it.

8.

Continue with

“Analyzing the Pure Dye Data” on page 29

.

-20 to -25

°C

Notes

28




Analyzing the Pure Dye Data

When you analyze the pure dye data, the SDS software extracts the calibration values from the pure dye plate document, then displays the spectral data in the Pure Dye Wizard.

The purpose of viewing the data in the Pure Dye

Wizard is to eliminate irregular pure dye peaks from the data set. The wizard presents the spectral data in sets of three wells, each containing the same pure dye.

Because the wells displayed by the wizard contain the pure dye at an identical concentration, the signal peaks for the set should be identical. Occasionally, pipetting inaccuracies or contamination can cause a well signal to shift slightly. While viewing the data, you must eliminate any outlying peaks.

To analyze the pure dye data:

1.

In the pure dye plate document, select

Analysis Extract Pure Dye Wizard.

2.

Follow the instructions as explained in the Pure

Dye Wizard to extract the pure dye spectra.

When presented with each screen:

a.

Inspect the spectra for shifts in peak location.

b.

If the data set contains an outlying peak, eliminate it by clicking the check box of the associated well.

Note:

Dye spectra are generally acceptable if they peak at the same location as their group but diverge slightly at other wavelengths.

c.

Click when finished.

d.

Repeat steps a through c for all remaining wells until prompted with a message reporting the extraction of the pure dyes.

The software extracts the pure spectra and stores the data as a component of the calibration file.

3.

Click dye plate document.

Notes

2a . Peak shift

2c

2b . Click here to remove it


29

1



4.

Select File Close to close the pure dye plate document.

5.

If you are performing the pure dye calibration for a:

• Standard 384-well sample block – The pure dye calibration is complete.

• Standard 96-well or Fast 96-well sample block – Run the second pure dye plate by repeating the following procedures:

–

“Creating a Pure Dye Plate Document” on page 24

–

“Running a Pure Dye Plate or Pure Dye

TLDA” on page 27

–


• TaqMan Low Density Array – Run the second and third pure dye TLDAs by repeating the following procedures for each

TLDA:

–

“Creating a Pure Dye Plate Document” on page 24

–

“Running a Pure Dye Plate or Pure Dye

TLDA” on page 27

–


IMPORTANT!

You must calibrate the instrument for all pure dye plates or pure dye TLDAs provided in your calibration kit.

Notes

30




Troubleshooting Pure Dye Calibrations

Troubleshooting

Table

Table 1-2 Troubleshooting pure dye calibrations

Observation

Software will not extract pure dye data

Possible Cause

During plate setup, the wrong plate type was assigned to the plate document


Create and run a new pure dye plate document with the proper plate type setting

Raw data from pure dye calibration appears strange

(see below)

A background plate was not run before the pure dye plate or TLDA

Run a background plate, then run the pure dye plate or TLDA again

Pure dye plate or TLDA was loaded backwards

1. Verify the pure dye wavelengths are as expected.

2. Rerun the pure dye plate or TLDA.

1

Signals plateau (saturation) Intensity is set too high/low Call Applied Biosystems

Technical Support.

Signal is too low

(<10,000 FSU)

More than two outliers per dye in a single row

• Evaporation

• Contamination

Rerun the pure dye plate or

TLDA. If the problem persists, discard the pure dye plate or TLDA and run a new one.

Notes


31


Performing an Instrument Verification Run

Performing an Instrument Verification Run

Overview

When to Perform

Applied Biosystems recommends performing an instrument verification run:

• Every 6 months

• As needed to verify the function of the 7900HT Fast System

Purpose of the

Verification Run

An instrument verification run is used to verify the performance of the 7900HT Fast

System. During the run, TaqMan

®

RNase P reagents detect and quantify genomic copies of the human RNase P gene. (The RNase P gene is a single-copy gene encoding the

RNA moiety of the RNase P enzyme.)

For the 7900HT Fast System instrument verification runs, the TaqMan RNase P reagents are available in the following formats:

• TaqMan

®

RNase P Instrument Verification Plates (RNase P plates) – Verifies instrument performance for 7900HT instruments that use a Standard 384-Well

Block, Standard 96-Well Block, or Fast 96-Well Block.

• TaqMan

®

Low Density Array RNase P Kit (TLDA RNase P kit) – Verifies instrument performance for 7900HT instruments that use a TaqMan

®

Low Density

Array Block.

About RNase P

Plates

RNase P plates are pre-loaded with the reagents required to detect and quantify genomic copies of the human RNase P gene. Each well contains pre-loaded reaction mix

(TaqMan

®

Universal PCR Master Mix, RNase P primers, and FAM

™

dye-labeled probe) and a known concentration of human genomic DNA template.

Notes

32




The table below illustrates the arrangement of standards and samples on each type of

RNase P plate that is available for the 7900HT Fast System. The RNase P plates contain five replicate groups of standards (1250, 2500, 5000, 10,000, and 20,000 copies), two unknown populations (5000 and 10,000 copies), and template control (NTC) wells.

Sample Configuration RNase P Plate

TaqMan

®

RNase P 384-Well

Instrument Verification

Plate

1

Unknown 1

5000

Unknown 2

10000

TaqMan

®

RNase P

Instrument Verification

Plate

TaqMan

®

RNase P Fast 96-

Well Instrument Verification

Plate

Unknown 1

5000

NTC

STD 5000

STD 1250

STD 10000

Unknown 2

10000

STD 2500

STD 20000

Notes


33



About the TLDA

RNase P Kit

The TLDA RNase P Kit includes one empty TLDA and eight tubes of solution. Each tube of solution contains reaction mix (TaqMan

®

Universal PCR Master Mix, RNase P primers, and FAM

™

-MGB dye-labeled probe) and a known concentration of human genomic DNA template.

To perform an instrument verification run, you load each solution into the empty TLDA.

The figure below illustrates the arrangement of standards and samples. There are five replicate groups of standards (200, 400, 800, 1600, and 3200 copies), two unknown populations (800 and 1600 copies), and no template control (NTC).

M

N

K

L

O

P

D

E

F

G

A

B

C

H

I

J

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 PORT

NTC

1

Unknown A (800)

Unknown B (1600)

Standard 200

Standard 400

Standard 800

Standard 1600

Standard 3200

2

3

4

5

6

7

8

Notes

34





Time Required

2 hours

Materials

Required




Appropriate RNase P plate for your sample block:

• TaqMan

®

RNase P 384-Well Instrument

Verification Plate (PN 4323306)

• TaqMan

®

RNase P Instrument Verification

Plate (PN 4310982, standard 96-well reaction plate)

• TaqMan

®

RNase P Fast 96-Well

Instrument Verification Plate (PN 4351979)




TaqMan

®

RNase P 384-Well Instrument Verification Plate

Caution, May cause eye, skin, and respiratory tract irritation. Read MSDS.

Part Number 4323306



Not for use in diagnostic procedures

TaqMan

®

Low Density Array RNase P Kit

(PN 4351468)

Spectral Calibrati

Part No: 0000001

1000002 Lot No:

Made in USA

7900HT

TaqMan

®

Density Emp

Low ty Array

0000001

Part No:

Lot No: 1000002

.


Not for use in diagnostic proc edures

1

Notes


35

Notes

36





µL)


µL)


TaqMan

®


Guide.)

TaqMan

®


®

Low Density


GR2170

Workflow

To perform an instrument verification run:

1.

Perform a pure dye calibration (see

page 17 ) before performing the instrument

verification run.

2.


and

36 ).

3.

Prepare the RNase P plate (see

page 37

) or RNase P TLDA (see

page 39 ).

4.


5.

Run the RNase P plate or RNase P TLDA (see

page 43 ).

6.

Analyze the run data (see page 44 ).

7.

Verify instrument performance (see

page 47

).




Preparing the RNase P Plate

IMPORTANT!

Wear powder-free gloves when you handle the RNase plate.

1.

Retrieve the RNase P package from the freezer, then remove the packaged RNase P plate.

Note:

For the standard 96-well RNase P plate, also use the compression pad provided in the package. Compression pads are not provided and should not be used for the Fast 96-well or standard 384-well RNase P plates.

2.

Allow the RNase P plate to warm to room temperature (approximately 5 min).

3.

Remove the RNase P plate from its packaging.

Ambient >00:05:00

1

4.

Briefly centrifuge the RNase P plate in a centrifuge with plate adapter.

<1500

×g

00:00:05

Notes


37



5.

Verify that the liquid in each well of the RNase P plate is positioned at the bottom of the well. If not, centrifuge the plate again at a higher rpm and for a longer period of time.


6.

Proceed to

“Creating a Plate Document for the

Verification Run” on page 41

.

IMPORTANT!

To prevent degradation of the reaction components, start the run as soon as possible.



Notes

38




Preparing the RNase P TLDA

IMPORTANT!

Wear powder-free gloves when performing this procedure.

1.

Remove the TLDA RNase P kit from the freezer, then unpack the contents:

• Eight tubes of solution

• One empty TLDA

2.

Remove the empty TLDA from its packaging.

Verification reagents box

Empty

TLDA

1

3.

Allow the eight tubes of solution to thaw at room temperature (approximately 5 min).

4.

Mix the contents of each tube by tapping gently.

5.

Using the empty TLDA, load 100

µL of the first solution into the designated reservoir.

Note:

The insert in the TLDA RNase P kit describes the designated reservoirs.

Note:

TaqMan

Guide.


®


6.

Repeat step 5 for the remaining solutions.

Ambient >00:05:00

Notes


39



7.

Centrifuge and seal the three RNase P TLDAs.

Note:


Started Guide.

®


8.

Proceed to

“Creating a Plate Document for the

Verification Run” on page 41

.

IMPORTANT!

To prevent degradation of the reaction components, start the run as soon as possible.

Notes

40




Creating a Plate Document for the Verification Run

1.




2.



Note:



3.

Click

4.


a.

Assay – Select Standard Curve (AQ).

b.


c.

Template – Select the appropriate template, as described in the table below.




Sample Block)


Block)


Density Array


384 Well RNaseP Install Plate.sdt

96 Well RNaseP Install Plate.sdt

Fast 96 Well RNaseP Install.sdt


RNaseP.sdt

d.

If the RNase P plate or RNase P TLDA is labeled with a barcode, click the Barcode field, then type in or scan the barcode number.

e.

Click . The software creates and opens a plate document.

IMPORTANT!

Do not modify the plate document. The plate document template is programmed with the appropriate detector and method information for the instrument verification run.

4d

4e

4a

4b

4c

Notes


41

1



5.


a.


Save As dialog box.

b.




c.





Sample Block)


Block)


Density Array

Type...

Verification_<date in MMDDYY

format>


Verification_053105.

Verification_<date in MMDDYY

format>


Verification_053105.

FastVerification_<date in MMDDYY

format>

For example, the file name for a Fast plate run on May 31, 2005, would be:

FastVerifiation_053105.

Verification<date in MMDDYY

format>_TLDA

For example, the file name for an

RNase P TLDA run on May 31, 2005, would be: Verification_053105_TLDA.

d.


e.

Click document.


6.

Continue with

“Running the RNase P Plate or

RNase P TLDA” on page 43 .

5b . This should be

SDS Documents

5c

5e

5d

Notes

42




Running the RNase P Plate or RNase P TLDA

1.

In the plate document, select the


2.


1

3.

Place the RNase P plate or RNase P TLDA into the instrument tray as shown.

IMPORTANT!

The RNase P plate or TLDA must be oriented so that the A1 position matches the

A1 label on the instrument tray.

2


Well A1

1

Barcode

4.

Click . the IN position and performs the instrument verification run.

Note:


5.

When the instrument verification run is complete and the Run Complete dialog box appears, click to close the dialog box.

Notes


43



6.

Click , then remove the RNase P plate or RNase P TLDA from the instrument tray.

PHYSICAL INJURY


Before removing the RNase P plate or RNase P

TLDA, be sure to wait until the sample block reaches room temperature.

7.

Proceed to

“Analyzing the Instrument

Verification Run” below.

Analyzing the Instrument Verification Run

1.

Click (or select Analysis > Analyze). The

SDS software analyzes the run data. A status bar at the bottom of the plate document window indicates progress; the status bar disappears when the analysis is complete.

2.

View the results:

a.

In the Plate Grid, click the box above the A label to select all wells.

Click here

b.

Click the Results tab to view the plots.

3.

Choose from the following:

• Automatic Ct – The SDS software automatically generates baseline values for each well and threshold values for each detector.

Notes

44




• Manual Ct – The SDS software calculates baseline and threshold values for a detector based on the assumption that the data exhibits typical amplification curves. If you are setting the baseline manually, specify the Start and End cycles.

IMPORTANT!

After analysis, you must verify that the baseline and threshold were called correctly for each well by clicking on the well in the Plate Grid and viewing the resulting plots. For more information about manually adjusting the baseline and threshold settings, refer to the Sequence Detection Systems Software version 2.3

Online Help (SDS Online Help).

4.

If necessary, remove outliers:

a.

Determine the outlier well positions.

Note:

The C

T

vs. Well Position view in the Results tab can help you determine the outlier well positions. To access this view, click the Plot drop-down list at the bottom of the Amplification Plot.

1

4a

Notes


45



b.

In the Plate Grid, select the outlier wells.

Note:

To select more than one well at a time, hold down the ctrl key while selecting the wells.

The number of wells that can be removed to pass specifications is shown in the table below.

Format

Maximum No. of Wells that Can be

Removed from Each Set

Unknowns

6

Standards

1 Standard 96-well

RNase P Plate

Fast 96-well RNase

P Plate

Standard 384-well

RNase P Plate

RNase P TLDA

6

10

4

1

2

4

c.

Click the Setup tab, then select the Omit Wells(s) check box. A red

✕

appears in each of the selected wells in the Plate Grid.

5.

Click (or select Analysis > Analyze) to reanalyze the data.

6.

Proceed to

“Verifying Instrument Performance” on page 47

.

Notes

46




Verifying Instrument Performance

Specification

Calculation

During analysis, the SDS software generates a standard curve from the averaged threshold cycle (C

T

) values of the replicate groups of standards, then calculates the concentration of the two unknown populations using the standard curve. To complete the verification, the average copy number (mean quantity) and standard deviation for the unknown populations are entered into the following formula to assess the instrument performance:

(Qty Unk

(high copy)

)

− 3 (Qty σ

(high copy)

) > (Qty Unk

(low copy)

) + 3 (Qty

σ

(low copy)

)

where:

Calculation

Term

Value

Standard 96well RNase P

Plate

10,000

Unknown Populations

Fast 96-well

RNase P Plate

10,000

Standard 384well RNase P

Plate

10,000

RNase P TLDA

1600 Qty Unk

(high copy)

Qty

Qty Unk

Qty

σ

σ

(high copy)

(low copy)

(high copy)

Average quantity of high copy number

Quantity standard deviation of high copy number

Average quantity of low copy number

Quantity standard deviation of low copy number

5000 5000 5000 800

The calculation states that the average copy number of the larger (10,000 for reaction plates or 1600 for TLDAs) unknown population minus three standard deviations is greater than the average copy number of the smaller (5000 for reaction plates or 800 for

TLDAs) unknown population plus three standard deviations. If the calculation is true, then the instrument passes the validation specification, verifying instrument performance.

1

Notes


47



Calculating the

Results

1.

In the Results Table of the plate document window, locate and record the average copy number (Qty mean) and standard deviation (Qty std dev) for the two unknown populations.

1

2.

Insert the values into the specification calculation:

(Qty Unk

(high copy)

)

− 3 (Qty σ

(high copy)

) > (Qty Unk

(low copy)

) + 3 (Qty

σ

(low copy)

)

3.

Determine whether the specification calculation is true or false.

• If the calculation is true, the 7900HT Fast System passes the validation specification. Instrument performance is verified.

• If the calculation is false, the 7900HT Fast System fails the validation specification. The instrument performance cannot be verified.

Notes

48




1

Notes


49



Notes

50


Chapter 2

Maintaining the

7900HT Instrument Hardware

Performing

Calibration and



Instrument

Hardware

Maintaining the

Computer and

Software

Recommended

Maintenance Schedule

See page 52

Removing and

Installing a Sample Block

See page 53

Maintaining the

Automation

Accessory

Replacing the

Plate Adapter

See page 62

Troubleshooting

Chemistry and

Assay Runs


Notes


Decontaminating the

Sample Block

See page 65

51

2

Chapter 2 Maintaining the 7900HT Instrument Hardware

Recommended Maintenance Schedule

Recommended Maintenance Schedule

To ensure optimal performance of your 7900HT Fast System, perform the following tasks as indicated in the table below.

Schedule

Weekly

Su M T W Th F S

Maintenance Task

• Cycle the computer and instrument power (power off, then power on the computer and instrument)

• Wipe instrument surfaces with a lint-free cloth

IMPORTANT!

Never use organic solvents to clean the 7900HT

Fast System.

Week (7 Days)

As needed

• Remove and install a sample block (see page 53

)

• Replace the plate adapter (see page 62

)

• Decontaminate the sample block (see

page 65 )

Notes

52



Removing and Installing a Sample Block

Removing and Installing a Sample Block

When to Perform

You need to remove/install a sample block when you:

• Decontaminate the wells of the sample block (see page 65 )

• Change sample blocks


Time Required

15 minutes. The time will be longer if you have to perform background or pure dye calibrations, or if you have an Automation Accessory.

Materials

Required


5/32 inch hex key

Note:

Some instruments require the 5/32inch hex key to remove the sample block locking bar. Other instruments have only a thumbscrew securing the sample block locking bar to the instrument chassis.

5/16 inch hex key

Note:

Some instruments may require a crescent wrench rather than the 5/16-inch hex key.

If you are replacing the sample block, one of the following:

• 7900HT System Standard 384-Well Block

• 7900HT System Standard 96-Well Block

• 7900HT System Fast 96-Well Block

• 7900HT System TaqMan

®

Low Density

Array Block

Warning

2

Notes

Workflow

To remove/install the sample block:

1.

Be sure all required upgrades to the SDS software and 7900HT instrument firmware are installed (see

page 83

).

IMPORTANT!

Failure to upgrade the software and firmware can make the instrument inoperable or result in damage to instrument components.

2.

Obtain the tools and materials required (shown above).

3.

Review the sample block handling information (see page 55 ).


53



4.

Remove the sample block (see page 56 ).

5.

Reinstall the same sample block or install a new sample block (see page 58 ).

6.

After the sample block is successfully installed, perform a background calibration

(see page 3 ) to verify that the sample block:

• Is connected and working properly

• Contains no contaminants that will interfere with fluorescent detection

IMPORTANT!

Perform a background calibration after installing the sample block, even if you are reinstalling the same sample block or replacing it with another block of the same format.

If you installed a new, uncalibrated sample block, also do the following:

7.

Change the plate adapter (see

page 62 ).

8.

Perform a pure dye calibration to create the pure dye calibration values for the new sample block (see

page 17 ).

9.

Perform a verification run to confirm the proper operation of the new sample block

(see page 32

).

If you are using an Automation Accessory, also do the following:

10.

If you changed sample block formats (for example, replacing a Standard 384-Well

Block with a Fast 96-Well Block), adjust the plate sensor switch on the Zymark

®

Twister Microplate Handler arm for the new plate format (see page 92

).

11.

Align the Plate Handler for the new plate format (see page 97 ).

12.

Align the fixed-position bar code reader for the new plate format (see

page 110

).

Notes

54




Handling the

Sample Block

The interchangeable sample blocks are delicate pieces of equipment containing several fragile components that can break if handled improperly. The figure below shows the correct locations for handling the interchangeable sample blocks.

Circuitry and connections to the instrument (Do Not Touch)

Heat sinks

2

(Bottom of module)

Hold sample block module from the sides

Notes


55



Removing the Sample Block

1.

Confirm the function of the current module:

Note:

If you do not have the SDS Automation

Controller software, skip to step 2

below.

a.

Double-click on the desktop (or select

Start > All Programs > Applied

Biosystems > SDS 2.3 > SDS Automation

Controller 2.3) to start the Automation

Controller Software.

b.

Select the Run Status tab. The module is operating normally if the software is receiving a temperature reading.

c.

Click Open/Close Door to rotate the instrument tray to the OUT position.

d.

Select File > Exit to close the Automation


2.

Power off and unplug the 7900HT instrument.

PHYSICAL HAZARD.

The instrument must be unplugged and powered off at all times during the following procedure.

Failure to comply can result in serious physical injury to the user or damage to the instrument.

3.

Wait 20 to 30 min for the heated cover to cool.


During instrument operation, the temperature of the sample block can be as high as 100 °C.

Before performing this procedure, wait until the sample block reaches room temperature.

4.

If the instrument tray is in the OUT position

(outside of the instrument), push it into the instrument to provide an open workspace.

Notes

56




5.

If using a Plate Handler, remove the covers for the fixed-position bar code reader and the underlying platform.

GR2009

GR2009

7900HT

Front view with Robot

7900HT FAST Real-Time PCR System

Fixed-position bar code reader and underlying platform covers

6.

Push the instrument tray inside the instrument, then remove the thermal cycler access cover to permit access to the sample block.

IMPORTANT!

The thermal cycler access cover is secured to the instrument by non-locking pins and may require force to remove it (no tools are required).

7.

Using a 5/16-inch hex key, turn the sample block locking bolt counter-clockwise until it is very loose but still attached to the sample block locking bar.

IMPORTANT!

Some instruments may require the use of an adjustable crescent wrench to loosen the sample block locking bolt.

8.

Loosen the thumbscrew securing the sample block locking bar to the instrument chassis (may be a 5/32-inch hex bolt on some instruments).

Access cover

Sample block locking bar

Sample block locking bolt thumbscrew

2

Notes


57



9.

Lift the sample block locking bar up and out of the instrument.

10.

Remove the sample block from the instrument:

a.

Rotate the release lever at the base of the sample block 90 degrees.

b.

Being careful not to damage the heat sinks on the bottom of the sample block, slide the sample block out of the instrument and place it on a clean, level surface.

Installing a Sample Block

IMPORTANT!

Before installing the sample block, perform all required upgrades to the SDS software and instrument firmware. Failure to upgrade the software and firmware can render the instrument inoperable or result in damage to instrument components.

1.

Load the sample block into the instrument compartment:

a.

Being careful not to damage the heat sinks on the bottom of the sample block, rest the sample block on the metal runners on either side of the instrument bay.

Notes

58




b.

Carefully slide the sample block into the instrument until the front of the block is flush with the rear of the locking bar.

c.

After it is seated, firmly press on the sample block to ensure a good connection.

2.

Replace the sample block locking bar.

3.

Tighten the thumbscrew (from step 8 on page 57 ) to secure the sample block locking bar to the instrument chassis (may be a 5/32-inch hex bolt).

4.

Using the 5/16-inch hex key, turn the sample block locking bolt clockwise until it is flush with the locking bar.

5.

Again, press on the right and left sides of the front surface of the sample block to ensure that it is seated securely.

6.

Replace the thermal cycler access cover:

a.

Fit the lip at the bottom of the access cover over the lower edge of the bay.

b.

Push the cover towards the instrument until it snaps into place.

IMPORTANT!

You must reinstall the thermal cycler access cover before powering on the instrument. Failure to do so prevents power from being applied to the thermal cycler electronics, heated clamp motor, and laser safety interlocks.

Notes


59

2



7.

If using a Plate Handler, replace the covers for the fixed-position bar code reader and the underlying platform (removed in step 5 on page 57 ).


8.

Plug in and power on the 7900HT instrument.

9.

Confirm the function of the installed sample block:

Note:

If you do not have the SDS Automation

Controller software, skip this step.

a.






Notes

60




b.

Select the Run Status tab. The sample block is operating normally if the software is receiving a temperature reading.

Does the software display temperatures?

Yes

No

Then…

the installation is successful.

The presence of temperature readings confirm that the

7900HT instrument successfully established the connection to the new sample block. the 7900HT instrument is unable to establish communication with the new sample block.

To troubleshoot the problem:

1. Power off and unplug the 7900HT instrument.

2. Remove the thermal cycler access cover.

3. Press on the right and left sides of the front plate of the sample block to ensure that it is seated securely.

4. Reinstall the thermal cycler access cover.

5. Repeat step 8

and

step 9 on page 60 until you hear

a high-pitched tone confirming communication between the instrument and sample block.

2

Notes


61


Replacing the Plate Adapter

Replacing the Plate Adapter

When to Perform

Replace the 7900HT instrument plate adapter after:

• Changing the sample block format (for example, replacing a Standard 384-Well

Block with a Fast 96-Well Block)

Note:

The sample block must be used with a corresponding plate adapter (that is, both the sample block and the plate adapter must have the same plate format).


Time Required

5 to 10 minutes

Materials

Required


Flat-blade screwdriver

One of the following:

• 384-Well Plate Adapter

• 96-Well Plate Adapter

• Fast 96-Well Plate Adapter

• TaqMan

®

Low Density Array Adapter

Workflow To replace the plate adapter:

1.


2.

Replace the plate adapter (see page 63 ).

Notes

62




Replacing the Plate Adapter

1.

If the instrument tray is inside the 7900HT instrument, move the instrument tray to the OUT position:

a.



Biosystems > SDS 2.3 > SDS 2.3) to start the SDS software.

b.

Click (or select File > New).

c.

In the New Document dialog box, click .

d.

In the new plate document, select the

Instrument > Real-Time tabs.

e.

Click .

f.

The instrument tray rotates to the OUT position.

g.

Select File > Exit to close SDS software.

2.

Remove the four screws attaching the plate holder to the plate arm.

Unscrew

2

Unscrew

3.

Remove the plate adapter from the instrument tray.

Note:

If changing sample block formats (for example, replacing a Standard 384-Well Block with a Fast 96-Well Block), store the plate adapter with the sample block of the same format.

Notes


63



4.

Place the new plate adapter into the instrument tray with the A1 label in the rear-left corner.

IMPORTANT!

Make sure to install the correct version of the plate adapter for the plate format you intend to use. The plate adapters are labeled with the format they support.

5.

Replace and tighten the four screws in the order shown.

IMPORTANT!

The order in which the screws are tightened is important to ensure proper alignment of the plate to the sample block inside the

7900HT instrument.

3

2

Well A1

1

Label

4

Notes

64



Decontaminating the Sample Block

Decontaminating the Sample Block

Overview

Using Other

Methods

This section describes how to decontaminate the wells of a sample block. The procedure eliminates residual PCR-related products, including fluorescent labeled TaqMan

® probes.

IMPORTANT!

If you plan to use a decontamination method other than the one in this guide, check with Applied Biosystems first to ensure that the method will not damage the sample block or the 7900HT instrument.

When to Perform

Decontaminate the sample block as often as needed.

Note:

Decontamination is generally performed to resolve problematic background calibrations, where one or more wells consistently exhibit abnormally high signals indicating the presence of a fluorescent contaminant.


Time Required

30 minutes. The time may vary, depending on the extent of the contamination.

Materials

Required



2

Pipettors and pipet tips (100-µL)

10% Sodium hypochlorite (bleach) solution

Water, deionized

Isopropanol, 100 percent pure

Cotton swabs and lint-free cloths

Notes


65



Workflow

To decontaminate the sample block:

1.

Remove the sample block from the 7900HT instrument (see page 53 ).

2.

Obtain the tools and materials required for decontamination (shown above).

3.

Decontaminate the sample block:

• For a Standard 384-Well Block, Standard 96-Well Block, or Fast 96-Well

Block, see page 67

• For a TaqMan

®

Low Density Array Block, see page 70

4.

Replace the sample block (see page 58 ).

5.

Perform a background calibration to confirm that the contamination has been removed (see

page 3 ).

Notes

66




Decontaminating a Sample Block

This procedure applies to a:

• Standard 384-Well Block

• Standard 96-Well Block

• Fast 96-Well Block

1.

Suspected contaminated wells are those that give a signal >4000 FSU during a background

calibration (see page 14 ). Using the SDS

software, isolate the suspected contaminated

wells as explained on page 15

.

2.

Locate the suspected contaminated wells on the sample block, using the figure at right as a guide.

Warning

Circuitry and connections to the instrument

(Do Not Touch)

Well A-1

2

Notes


67



3.

Rinse (pipet and remove) each contaminated well with three treatments of deionized water at the appropriate volume for the sample block:

• 40

µL per well for a Standard 384-Well

Block

• 150


Block

• 55

µL per well for a Fast 96-Well Block

Note:

Absolute isopropanol can be substituted for water in the third treatment.

CHEMICAL HAZARD.

Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the

MSDS, and follow the handling instructions.

Wear appropriate protective eyewear, clothing, and gloves.

4.

Using a cotton swab, scrub inside of each contaminated well.

Note:

If you are decontaminating a Standard

384-Well Block, remove some of the cotton from the swab tip; this allows the swab to reach the bottom of the wells.

5.

Using a lint-free cloth, absorb the excess deionized water or isopropanol from the wells.

Notes

68




6.

Pipet the appropriate volume of 10% bleach solution into each suspected contaminated well:

• 40


Block

• 150


Block

• 55



Sodium hypochlorite (bleach) is a liquid disinfectant that can be corrosive to the skin and can cause skin depigmentation. Please read the



7.

Allow the sample block to sit for 3 to 5 min.

8.

Using a pipet, remove the bleach solution from the wells.

9.

Rinse (pipet and remove) the wells with deionized water at the appropriate volume for the sample block:

• 40


Block

• 150


Block

• 55


10.

Using a lint-free cloth, absorb the excess deionized water from the wells.

2

Notes


69



Decontaminating a TLDA Block

This procedure applies to the TaqMan Low Density

Array Block.

1.

Using a lint-free cloth, wipe the surface of the sample block (grey aluminum) with 10% bleach.


Sodium hypochlorite (bleach) is a liquid disinfectant that can be corrosive to the skin and can cause skin depigmentation. Please read the



2.

Wipe the sample block three times with distilled water.

3.

Wipe the sample block with isopropanol.


Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the



4.

Allow the sample block to air dry.

Notes

70


Chapter 3

Maintaining the Computer and

Software

Performing

Calibration and



Instrument

Hardware

Maintaining the

Computer and

Software

Maintaining the

Automation

Accessory

Recommended


See page 72

Archiving and Backing Up

SDS Software Files

See page 73

Defragmenting the

Hard Drive

See page 76

Managing

Local User Accounts in the SDS Software

See page 78

Updating the

SDS Software

See page 83

Troubleshooting

Chemistry and

Assay Runs

Updating the

Operating System Software

See page 83


Notes


Troubleshooting Software and Computer Problems

See page 84

71

3

Chapter 3 Maintaining the Computer and Software



To ensure optimal performance of your 7900HT Fast System, perform the following tasks as indicated in the table below.

Schedule

Weekly

Su M T W Th F S


Archive or backup SDS plate document files (see

page 73

)

Week (7 Days)

Monthly

Su M T W Th F S

Defragment the computer hard drive (see

page 76

)

Month (30 Days)

As needed

• Manage local user accounts in the SDS software (see page 78

)

• Update the SDS software (see

page 83 )

• Update the operating system software (see page 83 )

Notes

72



Archiving and Backing Up SDS Software Files

Archiving and Backing Up SDS Software Files

When to Perform

Weekly

Archiving SDS

Software Files

To conserve space on the computer hard drive, SDS software files can be archived using a data compression utility. A compression utility typically archives files by encoding the binary data they contain algorithmically, thereby reducing the size of a file.

Several commercially available compression utilities are available. PKZIP and *.arc are archive formats common to the Microsoft Windows

®

operating system.

Backing Up SDS

Software Files

Applied Biosystems strongly recommends that you back up the data generated by your

7900HT Fast System for two reasons:

• Backing up data protects against potential loss of data caused by an unforeseen failure of the computer or its hard drive(s).

• Backing up older data and removing it from the computer hard drive conserves space on the hard drive and optimizes performance.

Choosing a Backup Storage Device

Applied Biosystems recommends the use of one or more backup storage devices to prevent potential loss of data caused by unforeseen failures of the computer or its hard drive(s).

The CD-RW drive of the computer can serve as the backup storage device for your system. By saving your *.sds and *.sdt files to one or more writable CDs on a weekly basis, you can effectively backup the data generated by your 7900HT Fast System.

Developing a

Data

Management

Strategy

Applied Biosystems recommends developing a strategy for dealing with the files produced by the SDS software. During a single day of real-time operation, the

7900HT System can generate over 200 MB of data. Without a strategy for distributing and archiving SDS software files, the 7900HT instrument can easily fill the hard drive of the computer within just a few weeks of operation.

If you are performing real-time experiments on your 7900HT Fast System, check the amount of available space on your hard drive weekly. When the hard drive is within 20% of it’s capacity, transfer the older data to a backup storage device.

Note:

Real-time experiments include standard curve (AQ) assays and comparative C

(RQ) assays, as well as any amplification (AQ) runs you might perform on the

7900HT System for allelic discrimination assays or plus/minus assays.

T

To successfully manage the information produced by the 7900HT System, you should have a basic understanding of how data is collected, stored, and processed throughout the operation of the instrument. See

“Modes of Operation” on page 74

.

3

Notes


73



Modes of Operation

System Operation and Dataflow

Data management strategy is a crucial element of successfully integrating the Applied

Biosystems 7900HT Fast Real-Time PCR System into a laboratory workflow. During a single 24-hour period of real-time operation, the 7900HT instrument can produce more than 200 MB of data. To successfully manage the information produced by the

7900HT System, you should have a basic understanding of how data is collected, stored, and processed throughout the operation of the instrument.

Standard Modes of Operation

The 7900HT Fast System has two standard modes of operation, depending on the accessories purchased with the base instrument:

• Stand-Alone Operation (see below)

• Automated Operation (see page 75 )

Stand-Alone Operation

In this mode (the most basic configuration of the 7900HT Fast System), the 7900HT instrument is used without any additional components. In this configuration (see the figure below), a technician runs plates individually using the SDS software. The computer stores all data for instrument operation and provides the software for analyzing the run data.

GR2179

7900HT raven instrument front


Applied Biosystems 7900HT

Fast Real-Time PCR System

Instrument Firmware

3

Thermal Cycling and

Sequence Detection

4

Data Collection

Serial

Cable

Computer

SDS Software

1

2

Plate Document Used to Run a Plate

5

Raw Data Saved to the Plate Document

6

Plate Document

Created

Plate Document

Opened and Analyzed

Hard drive(s)

*.sds

file

*.sdt

file

Notes

74




GR2009

7900HT



GR2009

Applied Biosystems 7900HT

Fast Real-Time PCR System

Instrument Firmware

3

Thermal Cycling and

Sequence Detection

4

Data Collection

Automated Operation

In this mode, the 7900HT Fast System uses an Automation Accessory (Zymark

®

Twister

Microplate Handler and fixed-position bar code reader) to provide high-throughput operation suitable for small- to medium-scale studies. With this configuration (see the figure below), a technician can use the Automation Controller Software to run batches of plates unattended. As in Stand-Alone mode, the computer stores all data for instrument operation and provides the software for analyzing the run data.

Computer

SDS Software

1

Plate Documents

Created

Automation Controller

Software

2

Plate document files added to and run from the plate queue

5

Raw Data Saved to the Plate Documents

Hard drive(s)

*.sds

files

*.sdt

file

Serial

Cable

6

Plate Document

Opened and Analyzed

3

Notes


75


Defragmenting the Hard Drive

Defragmenting the Hard Drive

When to Perform

• At least once every month

• Before fragmentation reaches 10% (or when warned by the Windows operating system)

Purpose: Why

Defragment the

Hard Drive?

As the 7900HT Fast System is used and files are deleted and created, the free space on the computer hard drive eventually is split into increasingly smaller blocks.

Consequently, as the SDS software creates new files and extends old ones, the computer cannot store each file in a single block. Instead, the system will ‘fragment’ the files by scattering their component pieces across different sectors of the hard drive.

The fragmentation of SDS files decreases the performance of both the SDS software and the computer operating system. As the hard drive becomes fragmented, programs take greater time to access files because they must perform multiple seek operations to access the fragments. Defragmentation utilities defragment broken files by combining their component pieces at a single location on the hard drive, thereby optimizing system performance.

Notes

76


Defragmenting the Hard Drive

1.

In the Windows desktop, select

My Computer.

2.

In the My Computer window, right-click a hard drive and select Properties.


Defragmenting the Hard Drive

3.

In the Local Disk Properties dialog box:

a.

Select the Tools tab.

b.

Click to open the Disk

Defragmenter dialog box.

4.

Click .

3a

2

3b

3

4

5.

When the Defragmentation Complete dialog box displays, click .

6.

In the Local Disk Properties dialog box, click .

7.

Repeat steps 2 through 6 for the remaining drives on the computer.

Notes


77


Managing Local User Accounts in the SDS Software

Managing Local User Accounts in the SDS Software

Managing local user accounts includes the following:

• Enabling or disabling the SDS software login function (

page 79

)

• Adding user accounts ( page 79 )

• Editing user accounts ( page 81 )

• Deleting user accounts ( page 82

)

Non-Enterprise

Mode Only

This section applies to the SDS software in non-Enterprise mode (that is, the SDS software does not include the SDS Enterprise Database). For versions of the SDS software that do include the SDS Enterprise Database, user accounts are managed with the SDS User Account Manager software. For more information, see the SDS Enterprise

Database for the Applied Biosystems 7900HT Fast Real-Time PCR System

Administrators Guide.

When to Perform

As needed

Notes

78


Enable/Disable Login

Enabling the login function allows the SDS software to capture user information so that the system can be customized for each user (user preferences).

When the login is enabled, the Login dialog box appears when the SDS software is started.



To enable or disable the login:

1.




2.

Select Tools Options to open the Options dialog box.

3.

Click the General tab.

4.

Select or deselect the Enable login in non-

enterprise mode check box, as desired.

5.

Click OK to save the changes and close the dialog box.

4

3

Add a User Account

1.




5

3

Notes


79



2.

Select Tools Local User Account Manager to open the Local User Account Manager dialog box.

3.

Click Add User.

3

4.

Complete the Add Local User dialog box:

a.

User Name – Enter a user name. The user name must begin with a letter of the alphabet and cannot exceed 30 characters.

b.

Password/Confirm Password – Enter a password, then re-enter it to confirm. The password must begin with a letter of the alphabet and must be between 6 and 30 characters long.

Note:

Users can change their own passwords at a later time by selecting

Tools Change Password.

c.

Full Name pane – Enter the user’s First,

Middle, and Last names. The names cannot exceed 50 characters.

d.

User Group pane – Select an appropriate group for the user: Operator,

Administrator, or Scientist.

Note:

This information is not used for access control purposes.

e.

Status pane – Select an appropriate status for the user: Active or Disabled.

Note:

If a user has the Disabled status, the user cannot log into the software.

5.


Notes

80


4d

4e

4a

4b

4c

Edit a User Account

1.




2.


3.

Select the user account you want to edit, then click Edit User.



3

4.

Complete the Edit Local User dialog box:

a.

Full Name pane – Enter the user’s First,

Middle, and Last names. The names cannot exceed 50 characters.

b.

User Group pane – Select an appropriate group for the user: Operator,

Administrator, or Scientist.

Note:

This information is not used for access control purposes.

c.

Status pane – Select an appropriate status for the user: Active or Disabled.

Note:

If a user has the Disabled status, the user cannot log into the software.

Note:

You cannot edit a user’s name or password. Users can change their own passwords at a later time by selecting Tools Change

Password.

5.


Notes


4a

4b

4c

81

3



Delete a User Account

1.




2.


3.

Select the user account you want to delete, then click Delete User.

4.

At the prompt, click Yes. The user account is removed from the Local User Account Manager.

5.

Click Close to close the dialog box.

3

Notes

82



Updating the SDS Software

Updating the SDS Software

When to Perform

As needed

Upgrading the

SDS Software

Applied Biosystems continually develops the SDS software to provide increased functionality and reliability of the 7900HT Fast System. As updates become available,

Applied Biosystems sends notifications of the upgrades to all Applied Biosystems

7900HT Fast System customers.

Review all documentation accompanying the software upgrade (such as installation notes or user bulletin). The updated version of the software may contain new features that require special consideration.

Note:

Applied Biosystems service engineers perform regular SDS software updates during planned maintenance visits.

Reinstalling the

SDS Software

On rare occasions, when a piece of the SDS software becomes corrupt, it may be necessary to reinstall the software. In the event that the software must be reinstalled, observe the following guidelines:

• Unless instructed to do otherwise, remove the SDS software using the uninstall utility. Do not delete the program folder from the Program Files directory.

• Backup all data before reinstalling the SDS software.

• Reinstall the SDS software under a user login that has administrator privileges on the computer.

• Unless instructed to do otherwise, reinstall the SDS software to the same directory as the previous installation.

Updating the Operating System Software

When to Perform

Windows Service

Pack Updates

Do not upgrade the operating system of the computer connected to the 7900HT Fast

System unless instructed to do so by an Applied Biosystems representative. New versions of the Windows operating system can conflict with the SDS software and render the instrument inoperable.

If you want to install a service pack to update the operating system, check the release notes that are installed with the SDS software for compatibility issues.

Note:

Applied Biosystems service engineers maintain the operating system software as part of planned maintenance visits. During the visit, an engineer will update the computer operating system as upgrades become available and are validated by Applied

Biosystems.

3

Notes


83


Troubleshooting Software and Computer Problems

Troubleshooting Software and Computer Problems

Troubleshooting

Table

Table 3-1 Troubleshooting software and computer problems

Observation Possible Cause

SDS software will not start

The software crashes/freezes the computer or displays an error message

• Incorrect start-up sequence

• Corrupted software

• Computer hardware failure

• Operating System

(OS) corruption

• Loose bar code reader cable


Follow the solutions listed until the symptom goes away.

1

1. Power off the 7900HT instrument.

2. Check cable connections.

3. Restart the computer and log on to the computer.

4. Power on the 7900HT instrument.

5. Start the SDS software.

Communication error

Thermal cycler errors

Automation Controller

Software cannot find a plate document file

2

6. Restart the computer and log on to your computer.

7. Reinstall the SDS software.

8. Start the SDS software.

3

Contact Applied Biosystems

Service for OS problems or if the computer will not boot up at all. You may have to reload the

OS from the CDs.

4

Cables are connected incorrectly

Check cable connections and

COM port setup. See

Appendix B

on

page 153

.

Sample block module not fully engaged

Reseat the sample block module. See

“Removing and

Installing a Sample Block” on page 53

.

File not in correct location

Contact Dell for troubleshooting the computer hardware.

Remove file entry from plate queue and add the file to the plate queue again.

Notes

84





Observation

Dialog box does not respond to mouse clicks or key strokes

Possible Cause

Java Runtime Error


Click the close box of the dialog box to close it.

Run will not start

Computer is slow when analyzing data, opening or closing dialog boxes, and other software processes

No calibration file

No background data in calibration file

(background calibration has not been performed)

Perform background and pure dye calibrations.

See “Performing a Background

Calibration” on page 3 and

“Performing a Pure Dye

Calibration” on page 17

.

No pure dye data in calibration file

(pure dye run has not been performed)

Calibration file does not contain pure dye data for a dye used on the plate document

Calibration file was created on another instrument

Disk drive containing the plate document has less than 50 MB of free space

Check the capacity of the destination drive. If less than

50 MB of free space remains, remove or archive existing data files. See

“Archiving and

Backing Up SDS Software

Files” on page 73 .

Heated cover cannot reach running temperature because no plate loaded

Instrument tray contains a plate

Open the instrument tray and check that the instrument contains a plate.

Output stack contains a plate or plates

Remove all plates from the output stack of the Plate

Handler before starting the queue.

Hard drive is fragmented

Defragment the hard drive. See

“Defragmenting the Hard Drive” on page 76

.

Hard drive is almost full

Remove or archive existing

data files. See “Archiving and

Backing Up SDS Software

Files” on page 73 .

Notes


85

3




Observation

The computer will not logon to the Windows operating system

Possible Cause

Logon window does not appear


Restart the computer and log on to your computer.

You are not logged on as the Administrator

1. Log off of your computer.

2. Log on again as the

Administrator.

The computer will not boot up at all

After the above solutions have been tried, the problem is still not fixed

Cables are not connected or are not seated properly

Contact Dell for troubleshooting the computer hardware or OS.

After the above solution has been tried, the problem is still not fixed

Check the cables.

The boot disk is corrupted.

1. Boot directly off of the

Windows operating system installation CD.

2. Boot off of the emergency disk.

3. Reload the Windows operating system from the

CD.

Contact Dell for troubleshooting the computer hardware.

Notes

86


Chapter 4

Maintaining the Automation Accessory

Performing

Calibration and



Instrument

Hardware

Maintaining the

Computer and

Software

Maintaining the

Automation

Accessory

Recommended


See page 88

Automation Accessory

Components and

Stack Positions

See page 89

Cleaning and

Replacing Gripper

Finger Pads

See page 90

Adjusting the

Plate Sensor Switch

See page 92

Aligning the Plate Handler

See page 97

Troubleshooting

Chemistry and

Assay Runs


Notes


Aligning the Fixed-Position

Bar Code Reader

See page 110

Troubleshooting the

Automation Accessory

See page 117

87

4

Chapter 4 Maintaining the Automation Accessory



To ensure optimal performance of the Automation Accessory, perform the following tasks as indicated in the table below.

Schedule

Monthly

Su M T W Th F S


Inspect the gripper finger pads (see

page 90 )

Month (30 Days)

As needed

• Clean or replace the gripper finger pads (see page 90 )

• Adjust the plate sensor switch (see

page 92 )

• Align the Plate Handler (see page 97

)

• Align the fixed-position bar code reader

(see page 110 )

Notes

88



Automation Accessory Components and Stack Positions

Automation Accessory Components and Stack Positions

Automation

Accessory

Components

The Automation Accessory includes the Zymark

®

Twister Microplate Handler and the fixed-position bar code reader.

Refer to the figure below for the components discussed in this section.

(cross-sectional view of the gripper)

Plate-sensor switch

Gripper

Adjustment knob

Plate stack

Expansion stacks

Zymark Twister Microplate Handler

Fixed-position bar code reader

Plate Stack

Positions

The Zymark Twister Microplate Handler alignment is performed using the Zymark

®

Twister Software. The software refers to the positions of the plate stacks differently than the Automation Controller Software. The figure below lists the positions defined by the

Zymark Twister Software and the Automation Controller Software equivalents.

(front of instrument)

2

1

0

3

4

7

6

5

Bar code

Well A1

Zymark Twister

Software

Position 0

Position 1

Position 2

Position 3

Position 4

Automation

Controller

Software

Output

(unused)

Instrument

(unused)

Stack 1

4

Notes


89


Cleaning and Replacing Gripper Finger Pads

Cleaning and Replacing Gripper Finger Pads

When to Perform

The adhesive used to affix bar code labels to certain brands of microplates can build up on the gripper pads of the Zymark Twister Microplate Handler. Over time, the residue can cause the gripper pads to stick to the microplates while handling them, causing misfeeds.

To prevent buildup, Applied Biosystems recommends that you:

• Inspect the gripper pads monthly

• Clean or replace the pads as needed


Time Required

10 minutes

Materials

Required


Finger Pad Replacement Kit, containing

10 finger pads (PN 4315472)

Flat-blade screwdriver, small

Phillips head screwdriver, small

Isopropanol in a squeeze bottle

Workflow To clean and replace the gripper finger pads:

1.


2.

Clean (see below) or replace (see page 91 ) the gripper finger pads.

Notes

90



Cleaning and Replacing Gripper Finger Pads

Notes

Cleaning the

Finger Pads

1.

Wipe each pad thoroughly with Isopropanol until the residue has been removed.

CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the

MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Replacing the

Finger Pads

2.

If the pads appear rough or the adhesive cannot be removed, replace the pads as described on page 91 .

1.

Using a small Phillips-head screwdriver, remove the two small Phillips-head screws from the fingers on each side of the gripper, then remove the fingers.

Note:

screws.

Move the Plate Handler arm into any position where it is easy to access the

2.

Using a small flat-blade screwdriver, pry the worn finger pads off the fingers.

Note:

The manufacturer recommends replacing all finger pads at the same time.

3.

Clean any residual adhesive off the fingers using isopropanol.

CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the

MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

4.

Remove a replacement finger pad from the paper backing, then place the finger pad on the appropriate finger position.

5.

Repeat step 4 for the remaining finger pads.

6.

Install the fingers with the fingers pointing down and the finger pads toward the center of the gripper.

7.

Insert the screws into the fingers and tighten.

Note:

The screws do not automatically align the grippers. Make sure that the finger pads are making good contact with the plate when the Plate Handler arm grips a plate.

4


91


Adjusting the Plate Sensor Switch

Adjusting the Plate Sensor Switch

When to Perform

Adjust the plate sensor switch (located underneath the arm of the Plate Handler):

• After changing the sample block format (for example, replacing a Standard 384-

Well Block with a Standard 96-Well Block)

Note:

The Automation Accessory should not be used for running Fast 96-well reaction plates.

Note:

The various plate types have different dimensions, which can affect the way the Plate Handler grips the plates. Adjusting the plate sensor switch after changing sample block formats ensures smooth operation of the Automation Accessory.

• If the Plate Handler is having difficulty sensing plates


Time Required

20 minutes

Materials

Required


Appropriate plate type for your current sample block; one of the following:

• Standard 384-well reaction plate


• TaqMan

®

Low Density Array

Note:


Pictured: Standard 384-well reaction plate

Workflow To adjust the plate sensor switch:

1.


2.

Adjust the plate sensor switch (see page 93 ).

3.

Test the adjustment (see page 95 ).

4.

Confirm the Zymark Twister Software is closed (see

page 96 ).

Notes

92


Adjusting the Plate Sensor Switch

1.

Power off the Zymark Twister Microplate

Handler.


The Zymark Twister Microplate Handler must be powered off at all times during the following procedure. Failure to comply can result in physical injury to the user or damage to the Plate

Handler.

2.

Clear the switch position by turning the thumb wheel all the way to the Up extreme (as indicated on the side panel).



Thumb wheel

Plate-sensor switch

3.

Begin the adjustment of the sensor switch:

a.

Grasp a plate by the sides, making sure not to place pressure in the center of the plate to deform it.

b.

Place the plate between the fingers of the gripper assembly and align it to the middle of the centering device.

c.

While holding the plate in position, slowly turn the thumb wheel to lower the switch onto the plate until the switch:

• Contacts the top of the plate, and

• Emits a soft, audible “clicking” noise

IMPORTANT!

The sound emitted by the sensor switch is very faint and may be difficult to hear. To make the adjustment easier, place your ear close to the sensor switch while making the adjustment and listen for the switch to engage.

4

Notes


93

Notes

94



4.

Remove the plate and listen for the plate sensor switch to disengage.

Did you hear the switch disengage?

No

Then…

a. Move the switch Down a few steps by turning the thumb wheel in the direction indicated on the arm. b. Replace the plate inside the gripper and listen for the switch to engage:

– If you do not hear the switch engage, then remove the plate and repeat steps a and

b above.

– If you hear the switch engage, remove the plate and

continue to step a

below.

Yes a. Move the switch Up by turning the thumb wheel one step in the direction indicated on the arm. b. Replace the plate and listen for the switch to engage:

– If you hear the switch engage, remove the plate and repeat steps a and

b above.

– If you do not hear the switch engage, then you have successfully identified the zero point of the plate sensor switch.

Note:

At the zero point, one step of the thumb wheel in the

Down direction causes the switch to engage.

5.

After the zero point is established, carefully turn the thumb wheel in the Down direction the number of steps appropriate for your plate type, as indicated below:

Plate

Standard 384-well reaction plate

Standard 96-well reaction plate

TaqMan Low Density Array

Turn the thumb wheel in the Down direction…

15 steps

20 steps

15 steps

Note:

switch.

If you lose count, begin again from

step 4

and identify the zero point for the


Testing the Adjustment

1.

Place the plate in the input stack 1 of the Plate

Handler.

2.

Power on the 7900HT Fast System, the Plate

Handler, and the computer.

3.

Select Start > Programs > Zymark Twister

Plate Handler > Twister to start the Zymark

Twister Software.

4.

Click Manual Control to open the Manual

Control dialog box.

5.

Click stack 4. The Plate Handler arm moves over the input stack.



Click

6.

Click .

• If the adjustment was successful, the Plate

Handler arm will lower upon the plate until the plate sensor switch engages, confirming the presence of the plate.

• If the Plate Handler arm emits a grinding sound, adjust the plate sensor switch:

a.

In the Zymark Twister Software, click

to raise the Plate Handler arm.

b.

Turn the thumbscrew in the Down direction

10 steps.

c.

Repeat step 6 until the Plate Handler arm successfully detects the plate.

4

Notes


95

Notes

96



7.

Click , then click .

• If the adjustment was successful, the Plate Handler arm will grasp the plate and remove it from the plate stack.

• If the Plate Handler arm stops before the gripper fingers are able to contact the plate and fails to grasp or pick up the plate, adjust the plate sensor switch:

a.

Turn the thumbscrew in the Up direction 10 steps.

b.

Grasp the plate with one hand and, from the Zymark Twister Software, click

to release the plate.

c.

Replace the reaction plate into input stack 1 of the Plate Handler.

8.

Repeat steps 6 through 7 until the Plate Handler arm successfully retrieves the plate.

9.

Grasp the plate with one hand and, from the Zymark Twister Software, click to release the plate.

10.

Exit the Zymark Twister Software:

a.

Click .

b.

Click Quit Application to close the software.

Confirm the

Zymark Twister

Software Is

Closed

A defect in the Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed. Because the Zymark Twister Software conflicts with the SDS software, the residual elements of the Zymark Twister Software must be closed inside the Microsoft Windows

®

operating system Task Manager before continuing.

1.

Press the Crtl + Alt + Del keys in unison to open the Windows operating system

Security dialog box.

2.

C lick Task Manager to open the Windows operating system Task Manager dialog box.

3.

Click the Applications tab.

4.

Confirm that the software has closed by looking for the Zymark Twister Software entry in the Task list.

• If the software is completely closed, there is no entry for the Zymark Twister

Software.

• If the software is still running, click the Zymark Twister Software entry to select it, then click End Task.

5.

Close the Task Manager dialog box.



Aligning the Plate Handler

Aligning the Plate Handler

When to Perform

Align the Zymark Twister Microplate Handler if:

• You move the Applied Biosystems 7900HT Fast Real-Time PCR System

• The Plate Handler is misaligned

Symptoms that the Plate Handler is out of alignment include:

– Excessive downward movement of the Plate Handler arm (the arm grinds when grasping or releasing plates)

– The Plate Handler arm collides with the plate stacks

– The Plate Handler arm releases plates above the bottom of the plate stacks

– Plates tip or tilt when placed into the instrument tray by the Plate Handler arm


Time Required

30 minutes

Materials

Required





• TaqMan

®

Low Density Array

Note:



4

Notes


97



Workflow

To align the plate handler:

1.


2.

Prepare the 7900HT instrument (see page 99 ).

3.

Align input stack 1 (see page 100 ).

4.

Align the Plate Handler to the 7900HT instrument (see page 102 ).

5.

Recheck input stack 1 (see

page 104 ).

6.

Define the bottom of the stack (see

page 105

).

7.

Define the positions of the remaining stacks (see

page 107 ).

8.

Confirm the Zymark Twister Software is closed (see

page 108

).

Notes

98




Preparing the Instrument for the Alignment

1.

Remove the covers for the fixed-position bar code reader and the underlying platform.

2.

Loosen the three black thumbscrews on the platform connecting the 7900HT Fast System and the Plate Handler base.

GR2009

GR2009

7900HT




Black thumbscrews

3.

Power on the 7900HT Fast System, the Plate

Handler, and the computer.

4.

Move the instrument tray to the OUT position:

a.






Note:

If an error dialog box appears reading, Machine calibration values are

not valid. Please refer to documentation

for calibration process, click .

b.

Click Open/Close Door. The 7900HT Fast

System moves the instrument tray to the

OUT position.

c.



5.

Select Start > Programs > Zymark Twister

Plate Handler > Twister to start the Zymark

Twister Software.

6.

Click Manual Control to open the Manual

Control dialog box.

Notes


99

4



Aligning Input Stack 1 (Zymark Position 4)

The alignment of input stack 1 (position 4 in the

Zymark Twister Software) is the first step in the alignment procedure. This alignment provides the basis for aligning all subsequent stacks on the Plate

Handler.

1.

Place an empty plate into input stack 1 (Zymark position 4).

2.

In the Zymark Twister Software, click position 4.

The Plate Handler arm moves over the input stack.

3.

Using the Vertical Positioning commands, lower the Plate Handler arm until it is just above the stack. The Vertical Positioning commands provide five ways to move the Plate Handler arm:

• Move the slider for large increments.

• Click inside the slider bar to move the arm in 250-step increments.

• Click the lower arrow on the bar to move the arm in 50-step increments.

• Click the up or down arrows in the Vertical

Adjustment text box to move the arm in

1-step increments.

• Click the Vertical Adjustment text box, enter a value, then press Enter to move the arm into a specific location.

Notes

100

Click

Slider

Slider bar (250 steps per click)

Down arrow (50 steps per click)

Text box arrows (1 step per click) or manual entry


4.

Using the Rotary Adjustment arrows, adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered.

• To move the Plate Handler arm clockwise, click the up arrow.

• To move the Plate Handler arm counterclockwise, click the down arrow.



Up arrow (moves the arm clockwise)

Down arrow (moves the arm counter-clockwise)

5.

Using the Vertical Positioning commands, carefully lower the Plate Handler arm into the stack. Change the Rotary Adjustment value as needed to center the gripper inside the stack.

6.

After the gripper is centered inside the stack, click on the plate.

to lower the Plate Handler arm

7.

Confirm the following:

• The plate is in the middle of the gripper span

• The plate sensor switch is contacting the plate

• The gripper and plate do not contact the side of the stack

8.

Click . The gripper grips the plate between its fingers.

9.

Select . The Plate Handler raises the arm to its highest position. If the plate contacts the sides of the stack, readjust the rotary position of the Plate Handler arm until the plate moves freely in the stack.

Note:

Contact between the plate and the stack or all stacks may be unavoidable. However, try to minimize the contact as much as possible.

10.

Using the Vertical Positioning commands, raise and lower the Plate Handler arm several times to check the alignment.

Notes


101

4



11.

Lower the Plate Handler arm to the bottom of the plate stack, click , then click .

The software records the rotary position for the

Zymark position 4 (input stack 1).

12.

Click plate.

. The gripper releases the

Aligning the Plate Handler to the

Instrument Tray

The next step is to align the Plate Handler arm to the instrument tray (Zymark position 2). This alignment will ensure a smooth exchange between the Plate

Handler arm and the instrument tray during instrument operation.

1.

If not already present, place an empty plate into input stack 1 (Zymark position 4) and pick it up with the Plate Handler arm:

a.


b.

Click .

c.

Click .

2.

Click position 2. The Plate Handler arm moves over the instrument tray.

3.

Using the Vertical Positioning commands, lower the Plate Handler arm until it is approximately

1 cm above the instrument tray.

Click here

Slider

Slider bar (250 steps per click)

Down arrow (50 steps per click)

Text box arrows (1 step per click) or manual entry

Notes

102


4.

Using the Rotary Adjustment arrows, center the gripper and plate along the Y-axis of the instrument tray.



Up arrow (moves the arm clockwise)

Down arrow (moves the arm counter-clockwise)

H

I

E

F

G

A

B

C

D

M

N

O

J

K

L

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

5.

Center the gripper and plate along the X-axis of the instrument tray by sliding the Plate Handler and base towards or away from the 7900HT Fast

System.

I

J

K

F

G

H

L

M

N

O

P

A

B

C

D

E

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Center the plate

6.

Again using the Rotary Adjustment arrows, center the gripper and plate along the Y-axis of the instrument tray, as shown in

step 4 above.

7.

Using the Vertical Positioning commands, carefully lower the Plate Handler arm onto the instrument tray and confirm that the plate rests squarely inside it.

8.

Tighten the three black thumbscrews on the platform connecting the 7900HT Fast System and the Plate Handler.

Center the plate

Black thumbscrews

9.

Release the plate from the Plate Handler arm:

a.

Click .

b.

Click .

Notes


103

4



10.

Click onto the plate.

. The Plate Handler arm lowers

11.

Save the rotary and vertical offset information:

a.

Click , then click . The software records the rotary position for the plate drawer (Zymark position 2).

b.

Click , then click . The software records the vertical position for the plate drawer.

Rechecking the Input Stack 1

Now that the positions of the Plate Handler and instrument are fixed, the Plate Handler stacks can be aligned and the positional values recorded.

1.

If not already present, place an empty plate into input stack 1 (Zymark position 4).

2.


The Plate Handler arm moves over the input stack.

Click

3.

Using the Vertical Positioning commands, lower the Plate Handler arm until it is 1 cm above the stack and verify that it is centered on the stack. If necessary, center the stack using the Rotary

Adjustment arrows.

4.

Carefully lower the Plate Handler arm into the stack. Center the gripper as it moves down the stack by adjusting the Rotary Adjustment arrows if needed.

5.

After the Plate Handler arm is centered inside the stack, click lowers upon the plate.

. The Plate Handler arm

Notes

104




6.


• The plate is in the middle of the gripper span.

• The plate sensor switch is contacting the plate.

• The gripper does not contact the side of the stack.

7.

Click .

8.

Click . The Plate Handler raises the arm to its highest position. If the plate contacts the sides of the stack, re-adjust the rotary position of the Plate

Handler arm until the plate moves freely inside the stack.

Note:

Contact between the plate and the stack or all stacks may be unavoidable.

However, try to minimize the contact as much as possible.

9.

Click , then click . The software re-records the rotary position for input stack 1 (Zymark position 4).

10.

While holding the plate, click and remove the plate.

Defining the

Bottom of the

Stack

The Automation Controller Software requires a bottom position value for all stacks. This value is used to prevent the Plate Handler arm from colliding or grinding as it moves to the bottom of each stack.

1.

Remove all plates from the instrument and the Plate Handler arm.

2.

Place an empty plate into the output stack (Zymark position 0).

3.

In the Zymark Twister Software, click position 0. The Plate Handler arm moves over the output stack.

4.

Using the Vertical Positioning commands, lower the Plate Handler arm until it is just above the stack.

5.

Using the Rotary Adjustment arrows, adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered.

6.

Using the Vertical Positioning commands, carefully lower the Plate Handler arm into the stack. Change the Rotary Adjustment value as needed to center the gripper inside the stack.

7.

After the gripper is centered inside the stack, click arm lowers upon the plate.

. The Plate Handler

4

Notes


105

Notes

106



8.


• The plate is in the middle of the gripper span

• The plate sensor switch is contacting the plate

• The gripper does not contact the side of the stack

9.

Click . The gripper grips the plate between its fingers.

10.

Select . The Plate Handler raises the arm to its highest position. If the plate contacts the sides of the stack, readjust the rotary position of the Plate Handler arm until the plate moves freely in the stack.

Note:

Contact between the plate and the stack may be unavoidable. However, try to minimize the contact as much as possible.

11.

Using the Vertical Positioning commands, raise and lower Plate Handler arm several times to check the alignment.

12.

Lower the Plate Handler arm, click , then click . The software records the rotary position for position 0 (the output stack).

13.

Click the

14.

.

While holding the plate, click and remove the plate.

15.

In the Vertical Adjustment field, enter –3200, then press Enter. The Plate Handler lowers the arm to a position near the base of the output stack.

16.

Carefully lower the Plate Handler arm until it is approximately 1–2 mm from the bottom of the stack.

17.

Click , click , then record the number in the Vertical Adjustment field. The software records the vertical position for position 0 (the output stack).

18.

Click position.

. The Plate Handler raises the Plate Handler arm to its highest

19.

In the Vertical Adjustment field, enter the Vertical Offset value determined in step 17 , and press Enter. The Plate Handler lowers the Plate Handler arm to a

Vertical Offset position.

20.

If necessary, readjust the Vertical Offset value and repeat steps 18 through 19 until satisfied with the setting.




Defining the

Positions of the

Remaining Stacks

1.

Place an empty plate into input stack 2 (Zymark position 5).

2.

In the Zymark Twister Software, click position 5. The Plate Handler arm moves over the input stack.

3.

Using the Vertical Positioning commands, lower the Plate Handler arm until it is approximately 1 cm above the stack, then center it using the Rotary Adjustment arrows.

4.

Carefully lower the Plate Handler arm into the stack. Center the gripper as it moves down the stack by adjusting the Rotary Adjustment arrows as needed.

5.

After the Plate Handler arm is centered inside the stack, click

Plate Handler arm lowers upon the plate.

. The

6.


• The plate is in the middle of the gripper span.

• The plate sensor switch is contacting the plate.

• The gripper does not contact the side of the stack.

7.

Click .

8.

Click . The Plate Handler arm raises to its highest position. If the plate contacts the sides of the stack, readjust the rotary position of the Plate Handler arm until the plate moves freely in the stack.

Note:

Contact between the plate and the stack may be unavoidable. However, try to minimize the contact as much as possible.

9.

Using the Vertical Positioning commands, raise and lower Plate Handler arm several times to check the alignment.

10.

Click , and click

Zymark position 5 (input stack 2).

. The software records the rotary position for the

4

Notes


107



11.

Repeat steps 1 through 10 for input stacks 3 and

4 to define Rotary Offset values for the remaining positions 6 and 7.

Zymark position 7

(input stack 4)

Zymark position 6

(input stack 3)

12.

Exit the Zymark Twister Software:

a.

Click .

b.

Click Quit Application to close the software.

13.

Replace the covers for the fixed-position bar code reader and the underlying platform

(removed in step 1 on page 99 ).

Confirm the Zymark Twister Software Is

Closed

A defect in the Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed. Because the

Zymark Twister Software conflicts with the SDS software, the residual elements of the Zymark Twister

Software must be closed inside the Windows operating system Task Manager before continuing.

1.

Press the Crtl + Alt + Del keys in unison to open the Windows operating system Security dialog box.

2.

C lick Task Manager to open the Windows operating system Task Manager dialog box.

3.

Click the Applications tab.

GR2009

GR2009

7900HT




Notes

108


4.

Confirm that the software has closed by looking for the Zymark Twister Software entry in the

Task list.

• If the software is completely closed, there is no entry for the Zymark Twister Software.

• If the software is still running, click the

Zymark Twister Software entry to select it, then click End Task.

5.

Close the Task Manager dialog box.



Notes


109

4


Aligning the Fixed-Position Bar Code Reader

Aligning the Fixed-Position Bar Code Reader

Two Bar Code

Readers

Two bar code readers are available for the 7900HT Fast System:

• CLV Fixed-Position Bar Code Reader

• LD Fixed-Position Bar Code Reader

This section provides procedures for aligning both bar code readers.

When to Perform

The fixed-position bar code reader must be set so that it automatically scans the plate’s bar code as the plate is placed into the instrument tray by the Plate Handler.

Align the fixed-position bar code reader:

• Whenever the Automation Accessory platform is moved from its calibrated position

(for example, during Service visits)


Time Required

20 minutes

Materials

Required





• TaqMan

®

Low Density Array

Note:



Workflow To align the fixed-position bar code reader:

1.


2.

Prepare the 7900HT instrument (see page 111 ).

3.

Align the fixed-position bar code reader. Be sure to follow the right alignment procedure for your bar code reader:

•

“Aligning the CLV Fixed-Position Bar Code Reader” on page 112

, or

•

“Aligning the LD Fixed-Position Bar Code Reader” on page 114

Notes

110




Preparing the Instrument for the Alignment

1.

Remove the cover for the fixed-position bar code reader.

GR2009

GR2009

7900HT



Fixed-position bar code reader cover

2.

Power on the 7900HT Fast System and the computer.

3.

Move the instrument tray to the OUT position:

a.






Note:

If an error dialog box appears reading, Machine calibration values are

not valid. Please refer to documentation

for calibration process, click .

b.

Click Open/Close Door. The 7900HT Fast

System moves the instrument tray to the

OUT position.

c.



4.

Continue with:

•

“Aligning the CLV Fixed-Position

Bar Code Reader” on page 112

, or

•

“Aligning the LD Fixed-Position Bar Code

Reader” on page 114

4

Notes


111



Aligning the CLV Fixed-Position Bar Code

Reader

Note:

This procedure is for the CLV Fixed-Position

Bar Code Reader. If you are aligning the LD Fixed-

Position Bar Code Reader, see

page 114

.

IMPORTANT!

The instrument tray must be in the

OUT position to align the bar code reader.

1.

Place a plate with bar code onto the instrument tray.

IMPORTANT!

Orient the plate so that well A1 aligns to the A1 position of the instrument tray and that the bar code faces the fixed-position bar code reader.

2.

Select Start > All Programs > CLV Setup v

4.1 to start the CLV software.

3.

In the CLV Setup window:

a.

Click the Device drop-down list and select

CLV42x.

b.

dialog box.

3b

Well position A1

Bar code

3a

Notes

112


4.

In the Terminal dialog box, select Percent

Evaluation.



5.

Loosen the black positional adjustment knob on the fixed-position bar code reader, and position the scan head of the reader as far as possible from the plate while maintaining the orientation towards the bar code on the plate.

6.

While watching the Terminal dialog box, slowly adjust the orientation of the fixed-position bar code reader until the Terminal dialog box reads more than 20%.

Note:

The Percent Evaluation depends on how many lines hit the bar code label.

Note:

It may be helpful to briefly place a sheet of white paper in front of the plate bar code to view the area scanned by the laser.

7.

When satisfied with the alignment, tighten the black positional adjustment knob on the fixedposition bar code reader.

8.

Click Exit to close the Terminal dialog box, then close the CLV software.

9.

Replace the cover for the fixed-position bar code reader (removed in step 1 on page 111 ).

Notes


Scan head of the fixedposition bar code reader

Black positional adjustment knob

4

113



Aligning the LD Fixed-Position Bar Code

Reader

Note:

This procedure is for the LD Fixed-Position

Bar Code Reader. If you are aligning the CLV Fixed-

Position Bar Code Reader, see

page 112

.

IMPORTANT!

The instrument tray must be in the

OUT position to align the bar code reader.

1.

Place a plate with bar code onto the instrument tray.

IMPORTANT!

Orient the plate so that well A1 aligns to the A1 position of the instrument tray and that the bar code faces the fixed-position bar code reader.

2.

Select Start > All Programs > PSC Laser

Data > LDHOST to start the LDHOST software.

3.

Establish communication with the fixed-position bar code reader: a. Click

Edit

Configuration dialog box.

b.

dialog box.

c.

d.

In the Device Control dialog box, click

(Connect to Device). The Terminal dialog box displays the fixed-position bar code reader response.

Click message.

to close the information

The LD Host program communicates with the bar code reader and updates the Edit

Configuration dialog box with the current configuration settings.

Notes

114

Well position A1

Bar code


4.

Configure the software for the alignment:

a.

In the bottom of the Edit Configuration dialog box, locate and select the Op. Modes tab.

Note:

You may need to use the arrows located in the bottom of the dialog box to locate the Op. Modes tab.

b.

In the Operating modes selection pane of the Edit Configuration dialog box, click the

Mode drop-down list and select Test.

c.

In the Device Control dialog box, click

RAM to toggle to EEPROM mode, then click Send.



Op. Modes tab

Mode drop-down list

RAM button

d.

In the Confirm dialog box, click YES to save to EEPROM.

The fixed-position bar code reader begins a continuous repeating scan of the bar code. The software updates the Terminal dialog box every

0.5 sec indicating the percentage of accurate reads completed during the 0.5 sec interval.

5.

Loosen the black positional adjustment knob on the fixed-position bar code reader, and position the scan head of the reader as far as possible from the plate while maintaining the orientation towards the bar code on the plate.

Scan head of the fixedposition bar code reader

Black positional adjustment knob

4

Notes


115



6.

While watching the Terminal dialog box, slowly adjust the orientation of the fixed-position bar code reader until the percent successful reading displays the highest number possible.

Note:

It may be helpful to briefly place a sheet of white paper in front of the plate bar code to view the area scanned by the laser.

7.

When satisfied with the alignment, tighten the black positional adjustment knob on the fixedposition bar code reader.

8.

Restore the fixed-position bar code reader to normal operation:

a.

In the Operating modes selection pane of the Edit Configuration dialog box, click the

Mode drop-down list and select

Serial on Line.

b.

In the Device Control dialog box, confirm that EEPROM is still selected, then click

Send.

c.

In the New Decision dialog box, click YES to save to EEPROM.

The bar code reader stops scanning the plate bar code and resumes normal operation.

9.

Click (Exit) to close the LDHOST software.

10.

Replace the cover for the fixed-position bar code reader (removed in step 1 on page 111 ).

Percent successful reads

Mode drop-down list

Notes

116



Troubleshooting the Automation Accessory

Troubleshooting the Automation Accessory

Troubleshooting

Table

Table 4-2 Troubleshooting the Automation Accessory

Observation

Plate Handler emits grinding noise when picking up or putting down plates

Plate Handler arm contacts racks when retrieving or stacking plates

The Plate Handler arm releases plates awkwardly into the plate racks

Reaction plates tip or tilt when placed into the instrument tray by the Plate

Handler arm

Plate Handler fails to sense or grasp plates

Possible Cause

Vertical offset too low

Plate detector switch set too high

Plate Handler rotary offset is incorrect or vertical offset is too low

Plates stick to the gripper fingers of the Plate Handler arm

Plate Handler does not restack plates in original locations

Fixed-position bar code reader not reading plate bar codes


Re-align the Plate Handler as explained in

“Aligning the Plate

Handler” on page 97

.

Plate sensor switch not adjusted properly

Gripper pads on the fingers of the Plate Handler arm are worn or dirty

Gripper pads are worn or dirty

Restack when finished option not selected

Bar code reader is misaligned

Bar code reader is broken

Adjust the plate sensor switch as

explained in “Adjusting the Plate

Sensor Switch” on page 92

.

Change the gripper pads as explained in

“Cleaning and

Replacing Gripper Finger Pads” on page 90

.

Change the gripper pads as explained in

“Cleaning and

Replacing Gripper Finger Pads” on page 90

.

Configure the Automation

Controller Software to restack the plates. See the Sequence

Detection Systems Software

version 2.3 Online Help (SDS

Online Help).

Re-align the fixed-position bar code reader as explained in

“Aligning the Fixed-Position Bar

Code Reader” on page 110

.

4

Notes


117


Troubleshooting the Automation Accessory

Notes

118


Performing

Calibration and



Instrument

Hardware

Maintaining the

Computer and

Software

Maintaining the

Automation

Accessory

Chapter 5

Troubleshooting Chemistry and

Assay Runs

Low Precision or Irreproducibility

See page 120

Standard Curve (AQ) and Comparative CT (RQ)

Quantitative Assay Runs

See page 124

Troubleshooting

Chemistry and

Assay Runs

Allelic Discrimination and Plus/Minus

End-Point Assay Runs

See page 126

5


Notes


119

Chapter 5 Troubleshooting Chemistry and Assay Runs

Low Precision or Irreproducibility

Low Precision or Irreproducibility

Overview

There are various reasons why an assay run with the Applied Biosystems 7900HT Fast

Real-Time PCR System can have less than optimal precision. Factors that can affect precision are described in detail below.

Improper Threshold Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Imprecise Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Non-Optimized Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Incomplete Mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Splashing PCR Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Drops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Writing on the Reaction Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Fluorescent Contamination on the Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Contaminated Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Improper or Damaged Plastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Low Copy Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Use of Non-Applied Biosystems PCR Reagents . . . . . . . . . . . . . . . . . . . . . . . . . 123

Improper

Threshold Setting

The key to high-precision quantitative PCR is accurate detection of the exponential

(geometric) phase. The Applied Biosystems 7900HT Fast Real-Time PCR System typically delivers sufficient sensitivity so that at least three cycles of the geometric phase are visible, assuming reasonably optimized PCR conditions. The SDS software calculates a fixed signal intensity, called a threshold, that each signal generated from

PCR amplification must reach before it is recognized by the software as actual amplification. The calculated threshold is an approximation, and should be examined and modified as needed.

Modifying the Threshold

In a real-time document of the SDS software, the threshold can be modified via the

Amplification Plot view following analysis of the run data. See the Sequence Detection

Systems Software version 2.3 Online Help (SDS Online Help) for more information.

Notes

120




Imprecise

Pipetting

The calculated quantities of target nucleic acid are directly affected by how precisely the template or reagent volumes are added to the reaction mixes.

Using Master Reaction Mixes

Applied Biosystems highly recommends using a master reaction mixture. Mix all common components (including the same template) to a set of reactions together, then dispense the mix to the replicate wells of the plate. When making each master reaction mix, add 5–10% additional volume to compensate for pipetting losses.

Using Pipettors

Pipetting precision is also improved by:

• Calibrating and servicing the pipettors regularly

• Pipetting larger volumes

• Reducing the number of pipetting steps whenever possible

• Increasing the consistency of the pipetting method

Consult the manufacturer about the correct method of dispensing liquid volumes accurately from the pipettor. For example, some pipettors are designed to deliver the designated volume at the first plunger stop, so ‘blowing out’ the residue may cause error.

Also, before using a new pipettor tip to serially dispense a master reaction mix, wet the tip once by drawing up some of the master reaction mix and dispensing it back into the mix again.

Non-Optimized

Chemistry

Chemistries that have not been optimized may be susceptible to inconsistencies. To maximize precision and reaction efficiency, optimize the primer and probe concentrations of each individual assay used. Refer to the TaqMan

®

Universal PCR

Master Mix Protocol (PN 4304449) for specific information about optimizing probe and primer concentrations for TaqMan-related chemistries.

Note:

TaqMan

®

Custom TaqMan

Gene Expression Assays, TaqMan

®

®

SNP Genotyping Assays, and

SNP Genotyping Assays do not require optimization.

Air Bubbles

Air bubbles in the wells can refract and distort the fluorescent signals. Ideally, the reagents would be applied to the wells using a pipetting technique that does not form air bubbles. However, if a plate does contain air bubbles, they can usually be removed by swinging, tapping, or briefly centrifuging the reaction plate.

Splashing PCR

Reagents

If PCR reagents splash the undersides of the optical adhesive covers, the heat from the lid may bake the liquid to the cover and may distort the signal. If splashing occurs, briefly centrifuge the reaction plate to remove all traces of liquid from the caps.

Notes

Incomplete

Mixing

For maximum precision, the PCR master reaction mix must be uniform. After adding all reaction components to the mix, vortex the mix for 4–5 seconds before aliquoting it to the wells of the plate. Also vortex any dilutions performed during the assay.

5


121



Drops

Drops of reagents that cling to the sides of the wells may not contact the thermal cycler sample block and consequently may not amplify. If the drop slides into the mix during

PCR, then the amplified products will become diluted and the result will be less than replicate wells that did not have drops. Therefore, carefully monitor the reaction plate as it is being transferred into the thermal cycler or 7900HT Fast System. If you observe any drops, take steps to remove them, such as centrifugation.

Writing on the

Reaction Plates

Do not write on any surface of the optical plates or the optical adhesive covers. The fluorescent properties of the ink can potentially affect the fluorescence emission from the plate and alter the results. Instead, note the contents of each well on a sheet of paper, or on a printout of the sample setup.

Fluorescent

Contamination on the Plates

Many compounds found in laboratories are fluorescent. If they come into contact with certain optical surfaces, such as the optical adhesive covers, the fluorescent results may be affected. For example, it has been noted that the powder used to lubricate the insides of plastic gloves often contains fluorescent compounds. Use only powder-free gloves and do not needlessly touch the reaction plates or optical adhesive seals.

Errors

Human errors from time to time are inevitable, such as pipetting into the wrong well, or making a dilution mistake.

Human error can be reduced in the following ways:

• Perform the assay in a systematic fashion. For example, the pattern of sample positions should be simple (avoid putting gaps in the rows).

• When pipetting the master reaction mix, look directly down into the reaction plate so that you can verify the transfer of the solution.

• If adding a small-volume reagent, such as template, place the drop of liquid on the side of the well. Briefly tap or centrifuge the plate afterwards to bring the droplet down into the well.

• After all pipetting is complete, visually inspect all the wells to confirm the presence of the reagent drops. Tapping or centrifuging the reaction plate will cause all the drops to slide down into the wells simultaneously.

• When making serial dilutions, be sure to change the pipet tip after each dilution step.

• Visually inspect the liquid volumes being pipetted to verify that the volume is approximately correct. A common mistake is using the wrong pipettor volume setting (such as setting 20 µL instead of 2.0 µL).

• Visually inspect the volumes of the completed reactions, looking for any wells that have volumes that do not match those of the other wells.

Contaminated

Sample Block

Any material contaminating the sample block can affect the results. For example, mineral oil reduces thermal transfer. Residue from writing on reaction plates darkens the wells, absorbing light.

Notes

122




The sample blocks should be periodically inspected for cleanliness. Sample block contamination can be visualized by running a background plate and inspecting the

resulting background signal for aberrant peaks above 4000 FSU (see page 15 ). See

page 65

for instructions on decontaminating the sample block.

Improper or

Damaged Plastics

Applied Biosystems recommends that you use Applied Biosystems optical plates, optical adhesive covers, and optical flat caps with the 7900HT Fast System. The plastics that comprise the optical parts undergo special testing for the absence of fluorescent impurities. Optical plates are frosted to improve the degree and precision of light reflection. Bent, creased, or damaged plastics may adversely affect the transmission of fluorescent signal or prevent proper sealing of a well resulting in evaporation, change in sample volume, and altered PCR chemistry. Make sure to use the correct plastics and visually inspect each reaction plate before use.

Note:

See

Appendix C, “Parts and Consumables,” on page 157 for a list of compatible

consumables and reagents.

Low Copy

Templates

When amplifying samples that contain very low quantities of nucleic acid (generally less than 100 molecules), expect lowered precision due to the Poisson distribution and biochemical effects related to binding probabilities. Low copy templates are also more susceptible to losses due to non-specific adhesion to plastic wells, pipettor tips, etc. The addition of carrier to the sample, such as yeast tRNA or glycogen, can help prevent these losses, increasing the precision and sensitivity of the assay.

Use of Non-

Applied

Biosystems PCR

Reagents

The Applied Biosystems buffer contains an internal passive reference molecule

(ROX

™ dye), which acts as a normalization factor for fluorescent emissions detected in the samples.

IMPORTANT!

Non-Applied Biosystems PCR buffers may not contain the ROX passive reference dye. If running non-Applied Biosystems chemistry, be sure to set the passive reference for your experiment as explained below.

Setting the Passive Reference

1. Open the appropriate plate document in the SDS software.

2. Select the Setup tab.

3. From the Passive Reference drop-down list, select the appropriate passive reference dye.

5

Notes


123


Standard Curve (AQ) and Comparative CT (RQ) Quantitative Assay Runs

Select the appropriate passive reference

Passive Reference drop-down list

Standard Curve (AQ) and Comparative C

T

Assay Runs

(RQ) Quantitative

Troubleshooting

Analyzed Data

When faced with irregular data, you can use the SDS software to diagnose some chemistry- and instrument-related problems. The following table contains a summary of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems.

Raw Data Plot

The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) for the selected wells during each cycle of the real-time PCR.

What to look for:

• Signal tightness and uniformity – Do the raw spectra signals from replicate groups and controls exhibit similar spectral ‘profiles’? If not, the plate or sample block could be contaminated.

• Characteristic signal shape – Do the samples peak at the expected wavelengths?

For example, samples containing only FAM

™

dye-labeled TaqMan

® not produce raw fluorescence in the wavelength of a VIC

®

probes should

dye component. A signal present in wells that do not contain the dye could indicate that the sample, master mix, or well contains contaminants.

• Characteristic signal growth – As you drag the bar through the PCR cycles, do you observe growth as expected? Absent growth curves may indicate a pipetting error (for example, well lacks template) or no amplification.

• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal.

Notes

124



Standard Curve (AQ) and Comparative CT (RQ) Quantitative Assay Runs

Multicomponent

Plot

The Multicomponent Plot displays a plot of normalized multicomponent data from a single well of a real-time run. The plot displays the component dye signals that contribute to the composite signal for the well.


• Correct dyes displayed – Does the plot display all dyes as expected? The presence of an unexpected dye may be the result of an error in detector setup, such as assigning the wrong reporter or quencher dye.

• ROX dye fluorescence level – Does the ROX dye signal fluoresce below the reporter dyes? If not, the lack of reporter fluorescence may be caused by an absence of probe in the well (a pipetting error).

• Background fluorescence – Do all dyes fluoresce above the background? The

Background signal is a measure of ambient fluorescence. If a dye fails to fluoresce above the background, it is a strong indication that the well is missing probes labeled with the dye (well does not contain probe, PCR master mix, or both).

• MSE Level – The MSE (mean squared error) is a mathematical representation of how accurately the multicomponented data fits the raw data. The higher the MSE value, the greater the deviation between the multicomponented data and the raw data.

Amplification Plot

The Amplification plot displays data from real-time runs after signal normalization and

Multicomponent analysis. It contains the tools for setting the baseline and threshold cycle (C

T

) values for the run.


• Correct baseline and threshold settings – Are the baseline and threshold values set correctly?

Identify the components of the amplification curve and set the baseline so that the amplification curve growth begins at a cycle number that is greater than the highest baseline number.

Identify the components of the amplification curve and set the threshold so that it is:

– Above the background

– Below the plateaued and linear regions

– Within in the geometric phase of the amplification curve

IMPORTANT!

After analysis, you must verify that the baseline and threshold were called correctly for each well by clicking on the well in the Plate Grid and viewing the resulting plots. For more information about manually adjusting the baseline and threshold settings, refer to the SDS Online Help.

• Irregular amplification – Do all samples appear to have amplified normally? The three phases of the amplification curve should be clearly visible in each signal.

5

Notes


125


Allelic Discrimination and Plus/Minus End-Point Assay Runs

• Outlying amplification – When the run data is viewed in the C

T

vs. Well Position plot, do replicate wells amplify comparably? Wells producing C

T

values that differ significantly from the average for the associated replicate wells may be considered outliers.

If a plate produces non-uniformity between replicates, some samples on the plate could have evaporated. Check the seal of the optical adhesive cover for leaks.

Allelic Discrimination and Plus/Minus End-Point Assay Runs

Troubleshooting

Analyzed Data

When faced with irregular data, you can use the SDS software to diagnose some chemistry- and instrument-related problems. The following table contains a summary of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems.

Raw Data

The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) for the selected wells during each cycle of the PCR.

What to look for...

• Signal tightness and uniformity – Do the raw spectra signals from replicate groups and controls exhibit similar spectral ‘profiles’? If not, the plate or sample block could be contaminated.

• Characteristic signal shape – Do the samples peak at the expected wavelengths?

For example, samples containing only FAM dye-labeled TaqMan probes should not produce raw fluorescence in the peak wavelength of the VIC dye component. A signal present in wells that do not contain the dye could indicate that the sample, master mix, or well contains contaminants.

• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal.

Notes

126


Chapter 6

Flags and Filtering

Performing

Calibration and



Instrument

Hardware

Maintaining the

Computer and

Software

Maintaining the

Automation

Accessory

Troubleshooting

Chemistry and

Assay Runs


Notes


Using Flags

See page 128

Flags by Analysis

See page 130

Description of Flags

See page 132

127

6

Chapter 6 Flags and Filtering

Using Flags

Using Flags

Flags and

Troubleshooting

Version 2.3 of the Sequence Detection Systems (SDS) Software features a system of flags that provide the basis for algorithmically identifying and eliminating problematic data from plate document and RQ study analysis. The flags are application-specific metrics, where each evaluates the data for a specific quality that is consistent with a problem common to a supported analysis. The system is designed to allow you to automatically exclude questionable data, alert you to potential problems, and provide you with a starting point for investigation.

About Flags

Each flag is the result of a unique algorithmic test that evaluates a specific property of the plate document data. The software performs the tests in a specific sequence and completes the analysis of the plate document even if one or more wells is flagged. Every flag yields a pass/fail result that indicates the success of the associated test. If a flag is enabled, the software indicates the status of the test in the appropriate column of the Results Table, as follows:

• A

✔ for a well that is flagged (Fail)

• Blank for a well that is not flagged (Pass)

In the SDS Software, you can automatically flag results data to meet specified criteria. Flags are assay specific and are user configured or automatically assigned.

• Automatically assigned flags are not user configurable. These flags are displayed in the Results table and the QC Summary tab.

• User configured flags are set/defined by the user. These flags are also displayed in the Results table and QC Summary tab.

Flag Symbols

Following an analysis, the software summarizes the results of the flag metrics for each sample by displaying one of four symbols. These symbols appear in the Flag column of the Results Table and in the Plate Grid.

Symbol

2

1

Definition

The sample passed all applicable flag tests (Pass).

One or more possible problems exists for the associated sample (Fail).

Note:

The symbol displays the number of flags the sample failed.

Omitted Well: Well omitted automatically by the algorithm

Note:

The symbol displays the number of flags the sample failed.

Omitted Well: Well omitted manually by user

Note:

Applied Biosystems recommends examining all samples that are flagged.

Notes

128



Using Flags

Customizing Flags

Most flags are a measurement of a specific aspect of the sample data. A flag is produced when the measurement exceeds a threshold, some of which you can customize to refine your analyses. In these cases, the user-defined parameters for the flags appear in the

Analysis Settings dialog box for the applicable analysis type. See “Configuring Flags for

Use” on page 129 for more information on entering custom thresholds.

Notes for Using

Flags

• Flags appear in the results tab, the plate grid, and the QC Summary tab.

• Flags are warnings. The software does not stop the analysis if one or more samples fail the flag tests.

• Applied Biosystems recommends examining flagged results (

✔).

• Holding the cursor over a column header in the Results Table displays a tooltip that lists the full name of the column (the default names are often acronyms).

Configuring

Flags for Use

While viewing the desired plate document or relative quantitation (RQ) study:

1.

Click to display the Analysis Settings dialog box.

2.

In the Analysis Settings dialog box, select the Plate tab (or select the Study tab if you are configuring the settings for an RQ study).

3.

Using the table on page 130

as a reference, do the following for each row in the

Flag Condition and Omit Settings table:

a.

If you want the software to display the results of the flag in the Results Table, select the check box in the Flag Condition column.

b.

Do not change the qualifying statement in the Condition column.

c.

In the Flag Condition Value column, enter a value for the software to apply as the threshold or limit for the associated flag.

d.

If you want the software to omit the wells that fail the flag from the analysis, select the check box in the Omit column.

Notes


129

6


Flags By Analysis

4.

If you want the software to apply the current settings to all future plate documents of the same analysis type, select Save Settings As My Default.

5.

Click Apply to reanalyze the plate document or study without closing the Analysis

Settings dialog box, or click OK.

Flags By Analysis

Note:

The plus/minus plate documents do not feature a system of flags.

Flags for SDS Software Version 2.3

Flags

Abb.

Name

BAF

BPR

Baselining algorithm failed

Bad passive reference signal

CAF C

T

calculation algorithm failed

DCN The distance between cluster and

NTCs is

EAF

EW

Exponential region algorithm failed

A well is empty

FOS Fluorescence is off-scale

GBO Well is an outlier

HMD A well has missing data

HNS A well has a noise spike

HRN A well has high relative noise

HSD High standard deviation in replicate group

HW

LME

LPL

NAP

Hardy Weinberg value is

Large mean squared error is

Laser power is low during the run

Percentage of plate wells not amplified

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔ ✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

Relative

Quantitation

Plate

Document

Study

✔

✔

✔

✔

See

Page

132

132

133

133

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

✔

133

134

134

139

136

136

136

137

✔

✔

✔

✔

✔

✔

‡

‡

135

137

138

138

Notes

130


Flags for SDS Software Version 2.3

Flags

Abb.

Name

NAW

NOC

POU

SNS

A well is not amplified

Number of clusters is

Percentage of outliers is

The number of samples is

✔

✔

✔

✔

✔

TAF Thresholding region algorithm failed

✔

‡ Visible only when viewing study data using the Plate Centric Table Orientation.

✔

✔

✔


Flags By Analysis

Relative

Quantitation

Plate

Document

✔

Study

✔

See

Page

✔

138

139

139

139

140

Notes


131

6

Description of Flags

Baselining Algorithm Failed (BAF)

Description/

Function

This flag indicates that the algorithm was unable to determine the best fit baseline for the data set.

Note:

The Exponential Region, Baselining, Thesholding, and C compute the flags for the remaining algorithms in the sequence.

T

Calculation

Algorithms are performed in sequence. If one flag fails, then the SDS software does not

Troubleshooting 1.

Select the affected sample in the Results Table.

2.

Select the Results tab.

3.

In the Amplification Plot, review the amplification curves. If necessary, set the baseline and threshold manually for the affected data.

Bad Passive Reference (BPR)

Description/

Function

This flag indicates that the passive reference signal for the associated sample may:

• Be below normal levels

• Contain irregular fluctuations (non-uniform signal)


Confirm the results of the flag test:

a.


b.


c.

Click to display the Multicomponent Plot.

d.

In the Multicomponent Plot, review the passive reference signal for irregularities.

2.

If the passive reference signal does contain inconsistencies, determine the location of the failed well in the Results Table.

3.

If the plate or TLDA used to run the sample is available, verify that the well contains fluid.

Fluctuations in the passive reference signal can be caused by:

• An empty or leaking well

• Formation of bubbles in the well

Notes


Description of Flags

132




4.

If the passive reference signal does not exhibit inconsistencies or abnormal behavior, consider changing the analysis settings for the flag to be less sensitive.

C

T

Calculation Algorithm Failed (CAF)

Description/

Function

This flag indicates that the software could not calculate a C

T error.

because of a computational

Note:


T

Calculation




2.


3.


Distance Between Cluster and NTCs (DCN)

Description/

Function

This flag indicates that the associated sample is located close to the no template control

(NTC) cluster, and may not have amplified well. If active, the SDS software omits all samples that do not meet condition criteria.

Troubleshooting

Insufficient signal may be the result of poor PCR or an insufficient number of thermocycles.

Exponential Region Algorithm Failed (EAF)

Description/

Function

This flag indicates that the software could not identify the exponential region of the amplification curve.

Note:


T

Calculation




2.


6

Notes


133



3.


Empty Well (EW)

Description/

Function

This flag indicates that the software has determined the associated well to be empty because the well:

• Does not contain sample or reagents (primers and TaqMan

®

probes).

• Produced very low fluorescence (or none at all)


Confirm the results of the flag test:

a.


b.


c.


d.

In the Multicomponent Plot, verify that the signals are below normal level for the plate/study.

2.

In the Results Table, determine the location of the failed well.

3.

If the plate or TLDA used to run the sample is available, verify that the well contains fluid.

Fluorescence Off-Scale (FOS)

Description/

Function

This flag indicates that the associated sample produced fluorescence in one or more cycles that exceeds the maximum detectable range for the 7900HT instrument

(CCD saturation).

Note:

Offscale data can corrupt the results of a run if the offscale data is located at a cycle that interferes with the C

T

calculation.



2.


3.


4.

In the Raw Data and Multicomponent Plots, evaluate the effect of the offscale data.

Does the offscale data interfere with the C

T well from the analysis.

calculation? If so, consider omitting the

Notes

134




5.

If possible, determine the source of the offscale result.

A variety of factors can produce offscale data including:

• Sample Overload – High concentrations of sample can produce offscale data.

• Contamination – A fluorescent contaminant on the exterior a well or within the reaction can produce consistently high signals throughout a run.

Outlier Well (GBO)

Description/

Function

This flag indicates that the C

T

of the sample deviates significantly from those of the associated replicate group and has been identified as an outlier based on the Grubbs test

(also known as the extreme studentized deviate [ESD] method for outlier removal).

Note:

The algorithm does not remove samples that achieve C for the replicate group.

T s within 0.25 of the mean

Note:

If two sample produce identical C

T s but are identified as outliers, then the software flags both wells.


In the SDS RQ Manager Software, note the well positions of the outlier and associated replicates.

2.

Select Table Orientation Detector Centric.

3.

In the upper-left pane, select the detector that the outlier evaluates to view the data for the replicate group.

4.

Select the Amplification Plot tab, then select Data Ct vs. Well Position to view the clustering of the C

T s for the replicate group.

5.

Evaluate the outlier. Omit or restore the well as necessary.

Hardy Weinberg Value (HW)

Description/

Function

This flag identifies samples that belong to a population that does not match Hardy

Weinberg equilibrium.

Troubleshooting

The samples may trigger this flag as a result of genuine allelic disequilibrium in the associated population or because of several experimental and technical issues.

6

Notes


135



Has Missing Data (HMD)

Description/

Function

This flag indicates that the associated sample contains one or more cycles for which no spectral data is present.

Note:

Although rare, samples can contain missing data as the result of an interruption to the

7900HT instrument hardware or software during the run.



2.


3.


4.

In the Raw Data and Multicomponent Plots, observe the missing data.

Does the missing data interfere with the C

T well from the analysis.

calculation? If so, consider omitting the

Has Noise Spike (HNS)

Description/

Function

This flag indicates that the amplification curve contains one or more abnormal data points which are inconsistent with the rest of the curve.



2.


3.

In the Amplification Plot, review the amplification curves for the noise spike. If the noise spike has biased the baselining or C

T

calculations, consider omitting the well from the analysis or set the baseline and threshold manually for the affected data.

4.

If no noise spikes are present, change the analysis settings for HNS to be less sensitive.

High Relative Noise (HRN)

Description/

Function

This flag indicates that the associated sample failed the high relative noise test.

The algorithm determines the magnitude of the noise for each well based on the reporter dye signal. It also estimates the mean and standard deviation of the noise for the entire plate. The flag is evaluated by comparing the noise of the well to the plate, then determining whether or not it exceeds the threshold in the analysis settings.

Notes

136






2.


3.


4.

In the Multicomponent Plot, observe the signal corresponding to the background fluorescence. If necessary, omit the well.

5.

Change the analysis settings to increase or decrease sensitivity, as desired.

High Standard Deviation in Replicate Group (HSD)

Description/

Function

This flag indicates that the standard deviation of the C

T s for the replicate group exceeded the criteria defined in the “High standard deviation in a replicate group” row of the Study tab in the Analysis Settings dialog box.


In the SDS RQ Manager Software, note the well positions within the outlier and associated replicates.

2.

Select Table Orientation Detector Centric.

3.

In the upper-left pane, select the appropriate detector to view the relevant data for the replicate group.

4.

Select the Amplification Plot tab, then select Data Ct vs. Well Position to view the clustering of the C

T s for the replicate group.

5.

Evaluate the C

T

of the flagged well in relation to the replicates. Omit or restore the well as necessary.

Large Mean Squared Error (LME)

Description/

Function

This flag indicates that the associated sample failed the Mean Squared Error test. To perform the test, the software compares the maximum value for the mean squared error for the well to the criteria defined in the “Large Mean squared error is” row the Analysis

Settings dialog box. If the statement evaluates True, then the software flags the well.



2.


3.


6

Notes


137



4.

In the Multicomponent Plot, observe the MSE curve in the plot.

Factors that can produce a Large Mean Squared Error include:

• Improper Pure Dye Calibration

• Incorrect dyes specified in setup

• Offscale fluorescence

Laser Power Low (LPL)

Description/

Function

This flag indicates that the power supplied to the laser of the 7900HT instrument decreased below the acceptable level at least once during the associated run.

Note:

Applicable only to runs performed with version 2.3 and later of the SDS

Software.

Troubleshooting

Open the log file for the appropriate 7900HT Fast System and confirm the loss of power during the associated run.

IMPORTANT!

If the loss of laser power is consistent and is observed over multiple runs, an instrument component may be nearing the end of its life and require service or replacement.

Non-Amplified Plate (NAP)

Description/

Function

This flag indicates that the percentage of wells on the plate that did not amplify exceeded the threshold defined in the “Percentage of plate wells not amplified” row of the

Analysis Settings dialog box.

Note:

Empty wells are not included in the test.

Troubleshooting

Review the data of the wells that failed to amplify (flagged as NAW).

Not Amplified Well (NAW)

Description/

Function

This flag indicates that the associated sample failed to amplify. To determine this, the algorithm calculates the difference between the signal at the beginning of the run and the signal at the end of the run.

Applied Biosystems recommends using the default setting for the NAW calculation. To customize the setting, perform successive analyses of your data set using different settings and adjust the setting according to the desired sensitivity.

Notes

138






2.


3.

Observe the signals in the Amplification Plot. If necessary, omit the well.

Number of Clusters (NOC)

Description/

Function

This flag indicates that the number of clusters in the data set (excluding the NTC cluster) is outside the threshold range defined in the “Number of clusters is” row of the Analysis

Settings dialog box.



2.


3.

In the Allele Plot, observe the data point in relation to the related cluster. If necessary, omit the well.

Percentage of Outliers (POU)

Description/

Function

This flag indicates that the percentage of outliers on the plate exceeds the threshold defined in the “Percentage of outliers is” row of the Analysis Settings dialog box.

Small Number of Samples in Cluster (SNS)

Description/

Function

This flag indicates that the number of data points in the cluster is less than the threshold defined in the “The number of samples is” row of the Analysis Settings dialog box.

The software can flag wells as the result of:

• Low allele frequency

• Small sample population

• Outliers mistakenly identified as a cluster (one or two samples in a cluster may in fact be an outlier)

Troubleshooting

Review the calls and/or re-run the plate.

Notes


139

6



Thresholding Algorithm Failed (TAF)

Description/

Function

This flag indicates that the software could not calculate a threshold because of a computational error.

Note:


T

Calculation




2.


3.

In the Amplification Plot, review the amplification curves. If necessary, set the baseline and threshold manually for the affected run.

Notes

140


Appendix A

Adding Custom Dyes to the

Pure Dye Set

The Applied Biosystems 7900HT Fast Real-Time PCR System can be used to run assays designed with custom dyes (dyes not manufactured by Applied Biosystems). However, before using custom dyes with the 7900HT Fast System, you must create and run a pure dye plate made with the custom dyes. The purpose of the custom pure dye plate is similar to that of a standard pure dye plate: the SDS software uses the custom pure dye plate to create a spectral standard to distinguish the custom dye.

IMPORTANT!

To use a custom dye on your 7900HT Fast System, it must fluoresce within the spectral range measured by the instrument. The working range of the CCD camera is 500 to 660 nm.


Materials

Required



Reaction plates appropriate for your sample block:

• Standard 384-well reaction plates

• Standard 96-well reaction plates

• Fast 96-well reaction plates

Note:

You cannot create custom dyes for the TaqMan

®

Low Density Array Block.

Pipettors and pipet tips (100-µL)

Notes


141

Appendix A

Before You Begin

Water, deionized

Custom dyes

Tubes (2-mL and 10-mL)

Optical adhesive covers or Optical Caps

(PN 4323032)


Notes

142


Appendix A

Before You Begin

Workflow

To add custom dyes to the pure dye set:

1.


and

142

).

2.

Create a dilution series plate for each custom dye you want to add to the pure dye set (see

page 144 ).

3.

Create a plate document for the dilution series plate (see page 144 ).

4.

Run and analyze the dilution series plate (see

page 145 ).

5.

Create a pure dye plate with the custom dye(s) (see page 148

).

6.

Add the custom dye(s) to the SDS software (see

page 149

).

7.

Create a plate document template with the custom dye(s) (see

page 150 ).

Notes


143

Appendix A

Creating a Dilution Series Plate

Creating a Dilution Series Plate

1.

In the center wells of a reaction plate, prepare a dilution series of the custom dye (for example

25, 50, 100, 200, 400, 800, 1600, and 3200 nM concentration):

• For a standard 384-well or Fast 96-well reaction plate, use 20

µL per well

• For a standard 96-well reaction plate, use

50

µL per well

2.

Seal the wells of the reaction plate using an optical adhesive cover or Optical Caps

(PN 4323032).

IMPORTANT!

Do not use MicroAmp

®

caps

(domed) or Optical Tubes with the 7900HT Fast

System. You can use Optical Caps (PN 4323032)

(ONLY on the standard 96-well reaction plates) with the 7900HT Fast System.

3.

Repeat this procedure for each custom dye you wish to add to the pure dye set.

Creating a Plate Document for the Dilution Series Plate

1.




2.



Note:



3.

From the SDS software menu bar, click (or select File New).

Notes

144


Appendix A

Running and Analyzing the Dilution Series Plate

4.


a.

Assay – Select Allelic Discrimination.

b.


c.

Template – Select Blank Template.

d.

Barcode – Leave this field blank.

e.

Click . The software creates and opens a new plate document.

Note:

It is not necessary to configure detector, sample, or method information for the dilution series plate document. The purpose of the run is to establish the correct working concentration for the dye by viewing the intensity of the raw spectra produced by the wells in the dilution series.

5.

Repeat this procedure for each dilution series plate you created.

Running and Analyzing the Dilution Series Plate

1.

In the plate document, select the Instrument >

Plate Read tabs.

2.

Click Open/Close. The instrument tray rotates to the OUT position.

4d

4e

4a

4b

4c

Notes


145

Appendix A


3.

Place the prepared series dilution plate into the instrument tray as shown.

IMPORTANT!

The A1 position is located in the top-left side of the instrument.


Well A1

Barcode

4.

Click Post Read. The instrument tray rotates to the IN position and the instrument performs the run.

As the instrument performs the run, it displays status information in the Plate Read tab.

After the run, the status values and buttons are grayed-out, the Analysis button is enabled ( ), and a message indicates whether or not the run is successful.

5.

Click Open/Close to eject the reaction plate.

6.

Click software analyzes the raw run data.

7.

Click

Notes

146


8.

In the Raw Data Plot, determine the highest concentration of dye that does not produce a saturated signal, then record it for future use.

Saturated signals are characterized by their high peaks that rise beyond detectable levels

(> 65,000 fluorescent units) and appear as plateaus on the Raw Data plot.

The concentration of the custom dye that yields the highest possible signal but does not saturate is the maximum concentration for use with the

7900HT Fast System.

9.

Repeat this procedure for each dilution series plate you created.

Appendix A


Notes


147

Appendix A

Creating a Pure Dye Plate with the Custom Dye(s)

Creating a Pure Dye Plate with the Custom Dye(s)

1.

Prepare 5 mL of a custom dye at the concentration determined in step 8 on page 147 .

2.

Pipet the diluted custom dye to at least three columns of a reaction plate, as shown at right.

IMPORTANT!

The optical configuration of the

7900HT instrument requires that each custom dye occupy at least three columns of the reaction plate to permit adequate data collection.

• For a standard 384-well or Fast 96-well reaction plate, pipet 20

µL per well

• For a standard 96-well reaction plate, pipet

50

µL per well

3.

Repeat

steps 1 through 2 for each custom dye

you wish to add to the reaction plate.

4.

Seal the wells of the reaction plate using an optical adhesive cover or Optical Caps

(PN 4323032).

IMPORTANT!

Do not use MicroAmp

®

caps

(domed) or Optical Tubes with the 7900HT Fast

System. You can use Optical Caps (PN 4323032)

(ONLY on the standard 96-well reaction plates) with the 7900HT Fast System.

Notes

148


Appendix A

Adding the New Custom Dye(s) to the SDS Software

Adding the New Custom Dye(s) to the SDS Software

1.

From the SDS software menu bar, select

Tools > Dye Manager.

2.

In the Dye Manager dialog box, click .

3.

In the Add Dye dialog box:

a.

Enter a name for the custom dye.

b.

Click . The new dye appears in the

Dye Manager’s Custom dye list.

3a

3b

4.

Repeat

steps 1 through

3 for each custom dye you added to the custom pure dye plate

( page 148

).

5.

Click . The SDS software makes the new dye(s) available to pure dye plate documents.

Notes


149

Appendix A

Creating a Plate Document Template with the Custom Dye(s)

Creating a Plate Document Template with the Custom Dye(s)

1.

Click

2.


a.

Assay – Select Pure Spectra.

b.


c.

Template – Select Blank Template.

d.

Barcode – Leave this field blank.

e.

Click . The software creates and opens a new plate document.

2d

2e

2a

2b

2c

3.

Apply the new custom dye(s) to the plate document:

a.

Select the wells containing the first custom dye.

b.

Select the Setup tab.

c.

In the Dyes drop-down list, select the appropriate dye. The software applies the dye to the selected wells.

d.

Repeat steps a and b to configure the plate document with any additional custom dyes.

3b

3c

Custom dye added to selected wells of the plate document

Notes

150


Appendix A


4.

Save the custom pure dye plate document as a plate document template:

a.

Save As dialog box.

b.

In the Save in field, navigate to and open

AppliedBiosystems SDS2.3

Templates.

Note:

By saving the plate document template to the Templates directory, it becomes available from the Template dropdown list in the New Document dialog box.

c.

In the File name field, enter a name for the plate document template.

d.

Select SDS 7900HT Template Document

(*.sdt)

from the Files of type drop-down list.

e.

Click . The software saves the plate document as a plate document template.

5.

To run the custom dyes, see

“Performing a Pure

Dye Calibration” on page 17

.

4b . This should be

Templates

4c

4e

4d

Notes


151

Appendix A


Notes

152


Appendix B

Instrument Connections

Electrical

Connections

Figure B-1 and Table B-1 illustrate the electrical connections of the Applied Biosystems

7900HT Fast Real-Time PCR System components.

Power

H

Power

A

A B C

HI-POT

D

C

D

A

B

E

G

A B

Power

C

HI-POT

D

E

G

Power

C

Communications Cable

Power Cable

Figure B-1 Connections of the 7900HT Fast System

Table B-1 Connections of the 7900HT Fast System

Cable Type Connects… To…

A

B

C

D

E

G

‡

H

Communication

Comm/Power

Comm/Power

Computer (Monitor Port)

Computer (ISA Card 1)

Bar Code Reader Cable

‡See Figure B-2 on page B-154 .

Monitor

Mouse (not shown)

7900HT Instrument Serial Computer (Serial Port 1)

Comm/Power Computer (Keyboard Port) Hand-held Bar Code Reader

Communication Computer (Serial Port 2) Plate Handler (Port C)

Fixed-Position Bar Code Reader

Keyboard (not shown)

Notes


153

Appendix B

Fixed-Position

Bar Code Reader

Bar Code

Reader Cable

To Lava Card

(ISA Card 1)

Universal Voltage

Accessory Kit

(P/N 4334482)

G

Figure B-2 Fixed-position bar code reader connection

Power supply

Power

IEC 320 4-Position

Universal Power Strip

(PN 4333969)

Notes

154

IEC 320 North America

× (1)

IEC 320 Continental Europe

× (1)

Universal Jumper Cord set

× (4)

Jumper Cord set for Japan

× (4)

(see below for use in Japan)

IEC 320 U.K./Ireland

× (1)

IEC 320 Australia/

New Zealand

× (1)

IEC 320 Japan

× (1)

In Bag Marked “JAPAN ONLY”

To computer, monitor, hand-held bar code reader, and the Plate Handler

Note:

The order is not critical.

North America: 120V@15A MAX

Europe and all other locations: 250V@10A MAX

Figure B-3 Universal power strip

To install the Universal Voltage Accessory Kit (All Countries Except Japan):

1.

Connect one universal jumper to each accessory (see below for use in Japan).

2.

Connect the other end of the power jumpers to the power strip outputs.

3.

Choose the correct country specific power input cord for the geographical region and connect it to the power strip input.

4.

Power off all instrument accessories (monitor, computer, and Plate Handler).

5.

Connect the power-input cord to the AC outlet.


6.

Power on accessories.

7.

Discard unused cord sets.

Usage Guidelines for Japan

1.

Use only the cord sets supplied in the bag marked “JAPAN ONLY.”

2.

Discard all other unused cord sets.

Appendix B

Notes


155

Appendix B

Notes

156


Appendix C

Parts and Consumables

Note:

Part numbers listed in this appendix are for customers inside the United States.

Contact your Regional Sales Office for local Part numbers and prices. See “How to

Obtain Support” on page vii

.

Interchangeable Sample Block Modules and Accessories

The Applied Biosystems 7900HT Fast Real-Time PCR System features a Peltier-based, interchangeable sample block based on the technology established in the GeneAmp

®

PCR System 9700 thermal cycler.

The use of an interchangeable sample block:

• Reduces instrument downtime by allowing immediate replacement of the block.

• Permits easy access to the sample block for troubleshooting and maintenance.

• Supports multiple consumable formats.

• Provides several different modes of operation (including Max mode and programmable temperature ramps).

Table C-1 Sample block modules and accessories

Part No.

Description

4331406 7900HT System Standard 384-Well Block Upgrade Kit

Includes a Standard 384-Well Block, a 384-Well Plate Adapter, and a Sequence Detection Systems 384-Well Spectral Calibration

Kit (PN 4323977)

4331405 7900HT System Standard 96-Well Block Upgrade Kit

Includes a Standard 96-Well Block, a 96-Well Plate Adapter, and an ABI P

RISM

®

7900HT Sequence Detection Systems 96-Well


4351402 7900HT System Fast 96-Well Block Upgrade Kit

Includes a Fast 96-Well Block, a Fast 96-Well Plate Adapter, a

7900HT System Fast 96-Well Spectral Calibration Kit

(PN 4351653), a TaqMan

®

RNase P Fast 96-Well Instrument

Verification Plate (PN 4351979), and a TaqMan

®

Fast Reagents

Starter Kit (PN 4352407)

Quantity

1 kit

1 kit

1 kit

Notes


157

Appendix C

Consumables and Disposables

Table C-1 Sample block modules and accessories (continued)

Part No.

Description

4329012 7900HT System TaqMan

®

Low Density Array Upgrade

Includes a TaqMan

®

Low Density Array Block, microfluidic card sealer, centrifuge buckets and adapters, and a chemistry installation kit.

Quantity

1 kit

Consumables and Disposables

The 7900HT Fast System can run:

• 384-Well Optical Reaction Plates (also referred to as standard 384-well reaction

plates) sealed with optical adhesive covers

• MicroAmp

®

96-Well Optical Reaction Plates (also referred to as standard 96-well

reaction plates) sealed with optical adhesive covers or Optical Caps (PN 4323032, flat cap strips only)

• Optical 96-Well Fast Thermal Cycling Plates (also referred to as Fast 96-well

reaction plates) sealed with optical adhesive covers

• TaqMan

®

Low Density Arrays

The optical plates recommended above are designed specifically for fluorescence-based

PCR chemistries and are frosted to minimize external fluorescent contamination. Before running prepared optical plates on the 7900HT instrument, each plate must be sealed with the optical adhesive covers recommended above. Applied Biosystems optical adhesive covers are specifically designed to permit the transmission of light to and from the wells of the optical plate.

IMPORTANT!

Do not use MicroAmp

®

caps (domed) or Optical Tubes with the

7900HT Fast System. You can use Optical Caps (PN 4323032) (ONLY on the standard

96-well reaction plates) with the 7900HT Fast System.

Table C-2 Consumables and disposables for the 7900HT instrument

Part No.

Description

Optical Adhesive Covers

4313663 ABI P

RISM

®

Optical Adhesive Cover Starter Kit

Includes 20 ABI P

RISM

®

Optical Adhesive Covers, an Applicator, and an ABI P

RISM

®

Optical Cover Compression Pad.

4311971 ABI P

RISM

®

Optical Adhesive Covers

4360954 Optical Adhesive Covers

Quantity

20 Covers

100 Covers

25 Covers

Notes

158


Appendix C

Consumables and Disposables

Table C-2 Consumables and disposables for the 7900HT instrument (continued)

Part No.

4323032 Optical Caps, 8 Caps/Strip

Description Quantity

300 Strips/

Pkg

2400 Caps/

Pkg

Standard 384-Well Reaction Plates

4309849 384-Well Clear Optical Reaction Plate with Barcode (code 128)

4326270 384-Well Clear Optical Reaction Plate with Barcode (code 128),

10-Pack

Includes 10 of PN 4309849, 384-Well Clear Optical Reaction

Plates with Barcode

Standard 96-Well Reaction Plates

4306737 MicroAmp

®

96-Well Optical Reaction Plate with Barcode (code

128)

4326659 MicroAmp

®


128), 25-Pack

Includes 25 of PN 4306737, MicroAmp

®

96-Well Optical Reaction

Plates with Barcode

4314320 MicroAmp

®


128) and ABI P

RISM

®


Includes 100 ABI P

RISM

®

Optical Adhesive Covers (PN 4311971) and 5 of PN 4306737, MicroAmp

®

96-Well Optical Reaction Plates with Barcode

4312063 MicroAmp

®

Splash Free Support Base for 96-Well Reaction

Plates

Fast 96-Well Reaction Plates

4346906 Optical 96-Well Fast Thermal Cycling Plate with Barcode (code

128)

4312063 MicroAmp

®

Splash Free Support Base for 96-Well Reaction

Plates

TaqMan

®

Low Density Arrays

--TaqMan

®

Low Density Arrays

*

See the Applied Biosystems Web site.

50 Plates

500 Plates

20 Plates

500 Plates

100 Plates

100 Covers

10 Bases

20 Plates

10 Bases

Variable

*

Notes


159

Appendix C

Instrument Maintenance and Verification

Instrument Maintenance and Verification

The following sequence detection kits and reagents are used to perform routine maintenance on and verify the function of the 7900HT Fast System.

Table C-3 Consumables for calibration and verification runs

Part

Number

Description

Sequence Detection Systems Calibration Kits

4328639 ABI P

RISM

®

7900HT Sequence Detection Systems 96-Well

Spectral Calibration Kit

Includes three MicroAmp

®

96-Well Optical Reaction Plates: one preloaded and sealed background plate, and two preloaded and sealed pure dye plates containing eight separate dye standards

(FAM

™

, JOE

™

, NED

™

, ROX

™

, SYBR

®

Green, TAMRA

™

, TET

™

,

VIC

®

).

4323977 Sequence Detection Systems 384-Well Spectral Calibration Kit

Includes two 384-Well Optical Reaction Plates: one preloaded and sealed background plate and one preloaded and sealed pure dye plate containing eight separate dye standards (FAM, JOE, NED,

ROX, SYBR Green, TAMRA, TET, VIC).

4351653 7900HT System Fast 96-Well Spectral Calibration Kit

Includes three Optical 96-Well Fast Thermal Cycling Plates: one preloaded and sealed background plate, and two preloaded and sealed pure dye plates containing eight separate dye standards

(FAM

™

, JOE

™

, NED

™

, ROX

™

, SYBR

®

Green, TAMRA

™

, TET

™

,

VIC

®

).

4362745 TaqMan

®

Low Density Array Spectral Calibration Kit

Includes four empty TaqMan

®

Low Density Arrays (TLDAs) and four tubes: one background solution, one FAM

™

dye, one

ROX

™ dye, and one VIC

®

dye.

Instrument Verification Plates

4310982 TaqMan

®

RNase P Instrument Verification Plate

Includes one MicroAmp

®

96-Well Optical Reaction Plate. Each well contains preloaded reaction mix (master mix, RNase P primers, and FAM

™

dye-labeled probe) and template to detect and quantitate genomic copies of the human RNase P gene.

4323306 TaqMan

®

RNase P 384-Well Instrument Verification Plate

Includes one 384-Well Optical Reaction Plate. Each well contains preloaded reaction mix (master mix, RNase P primers, and FAM

™ dye-labeled probe) and template to detect and quantitate genomic copies of the human RNase P gene.

Quantity

3 x 96-Well

Plates

2 x

384-Well

Plates

3 x 96-Well

Plates

4 x TLDAs

1 x 96-Well

Plate

1 x 384-

Well Plate

Notes

160


Appendix C


Table C-3 Consumables for calibration and verification runs (continued)

Part

Number

Description

4351979 TaqMan

®

RNase P Fast 96-Well Instrument Verification Plate

Includes one Optical 96-Well Fast Thermal Cycling Plate. Each well contains preloaded reaction mix (master mix, RNase P primers, and FAM

™

dye-labeled probe) and template to detect and quantitate genomic copies of the human RNase P gene.

4351468 TaqMan

®

Low Density Array RNase P Kit

Includes one empty TaqMan

®

Low Density Array (TLDA) and eight tubes of solution. Each solution contains reaction mix (master mix,

RNase P primers, and FAM

™

-MGB dye-labeled probe) and template to detect and quantitate genomic copies of the human

RNase P gene.

Quantity

1 x 96-Well

Plate

1 TLDA

Notes


161

Appendix C


Notes

162


Index

A

absolute quantification troubleshooting

124

adjusting plate-sensor switch

92

to

96

adjustment knob

89

air bubbles

121

aligning

CLV fixed-position bar code reader

112

to

113

fixed-position bar code readers

110

LD fixed-position bar code reader

114

to

116

Plate Handler

97

to

108

amplification run starting

145

analyzing background data

14

pure dye data

29

Applied Biosystems contacting

vii

customer feedback on documentation

vii

Information Development department

vii

Technical Support

vii

assumptions for using this guide

v

Automation Accessory components

89

Plate Handler, See Plate Handler

See fixed-position bar code reader

B

background calibration about

3

,

17

analyzing the data

14

creating the plate document

10

preparing the plate

6

running the plate

12

when to perform

3

,

17

background component, about

3

background plate creating

8

preparing

6

running

12

background run troubleshooting

15

backing up SDS files

73

backup storage devices

73

7900HT Fast System Maintenance and Troubleshooting Guide bold text, when to use

v

C

calibrating the 7900HT instrument adjusting the plate-sensor switch

92

aligning the fixed-position bar code readers

110

aligning the Plate Handler

97

calibration background

3

,

17

CAUTION, description

vi

changing gripper finger pads

90

sample block module

53

chemistry non-optimized

121

troubleshooting

120

to

123

cleaning gripper finger pads

91

sample block wells

66

,

67

computer troubleshooting

84

consumables

Fast 96-well reaction plates

159

improper or damaged plastics

123


158

Optical Cap Strips

158

standard 384-well reaction plates

159

standard 96-well reaction plates

159

TaqMan Low Density Arrays

159

writing on reaction plates

122

contamination decontaminating the sample block

65

fluorescent, common sources

122

isolating on the sample block module

15

conventions bold text

v

for describing menu commands

v

IMPORTANTS!

vi

in this guide

v

italic text

v

Notes

v

user attention words

v

creating background plate

8

background plate document

10

custom pure dye plate documents

150

pure spectra plate document

24

163

Index

verification run plate document

41

custom pure dyes

141

customer feedback, on Applied Biosystems documents

vii

D

data management

73

decontaminating the sample block module

65

to

66

documentation, related

vi

F

finger pads cleaning

91

replacing

90

fixed-position bar code reader aligning CLV

112

to

113

aligning LD

114

to

116

connections

153

fluorescent contamination

122

detection system

153

G

gripper

89

H

hand-held bar code reader connections

153

I

imprecise pipetting

121

improper threshold setting

120

Information Development department, contacting

vii

installing plate adapter

62

sample block module

53

SDS software

83

SDS Software Updates

83

Windows service pack updates

83

instrument troubleshooting

84

instrument tray replacing the plate adapter

62

irreproducibility causes

120

to

123

italic text, when to use

v

L

LAVA software aligning the CLV fixed-position bar code

164

reader

112

to

113

launching

112

,

114

LDHOST software aligning the LD fixed-position bar code reader

114

to

116

low copy templates

123

M

maintenance schedule

52

,

72

,

88

calibration and verification runs

2

master mixes using

121

menu commands, conventions for describing

v

modes of operation

74

automated

75

stand-alone

74

MSDSs, obtaining

vi

,

vii

P

performing background calibration

6

pure dye run

21

pipetting errors

121

pipettors, using

121

plate adapter, changing

62

plate document background

10

pure spectra

24

verification run

41

Plate Handler

89

adjustment knob

89

aligning

97

to

108

cleaning the finger pads

91

gripper

89

plate stack positions

89

plate-sensor switch

89

replacing the finger pads

91

plate stacks positions

89

plate-sensor switch

89

adjusting

92

to

96

precision causes of low precision

120

to

123

preparing background plate

6

pure dye plates

21

RNase P plate

37

RNase P TLDA

39

process quality values (PQV) rules for

129

pure dye calibration analyzing the data

29

running the plate

27


Index

pure dye plate constructing for custom dyes

141

running

27

pure dye run troubleshooting

31

pure spectra calibration about

17

creating the plate document

24

preparing pure dye plates

21

R

reagents non-Applied Biosystems PCR reagents

123

TaqMan RNase P Instrument Verification

Plates

160

reinstalling the SDS software

83

relative quantification troubleshooting

124

replacing gripper finger pads

90

,

91

sample block module

53

RNase P plate running

43

RNase P TLDA running

43

RNase P verification run preparing the plate

37

preparing the RNase P TLDA

39

running background plate

12

pure dye plate

27

RNase P plate

43

RNase P TLDA

43

S

sample block locking bar

57

sample block locking bolt

57

sample block module cleaning sample block module wells

66

,

67

contamination

122

replacing

53

saturation, signal

147

SDS software installing

83

reinstalling

83

signal saturation

147

software, starting

10

,

24

,

41

,

56

,

60

,

63

,

79

,

81

,

82

,

99

,

111

,

144

spectral calibration, See pure dye runs

storage devices, backup

73

T

TaqMan Low Density Array RNase P Kit about

34

TaqMan RNase P Instrument Verification Plates about

32

analyzing

47

kits

160

Technical Support, contacting

vii

text conventions

v

threshold improper setting

120

TLDA RNase P card about

34

training, information on

vii

troubleshooting

7900HT instrument

84

background runs

15

chemistry problems

120

to

123

computer

84

end-point runs

126

fixed-position bar code reader

117

pure dye runs

31

real-time runs

124

SDS software

84

Zymark Twister Microplate Handler

117

U

updating

SDS Software

83

Windows operating system

83

user attention words, described

v

V

verification run creating the plate document

41

running the RNase P plate or RNase P TLDA

43

W

WARNING, description

vi

Windows service pack updates, installing

83

Z

Zymark Twister Software aligning the Plate Handler

97

to

108

testing the plate sensor switch

95


165

Index

166


Worldwide Sales and Support

Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents.

For sales office locations and technical support, please call our local office or refer to our

Web site at www.appliedbiosystems.com.

Appl ied Biosystems is committed to providing the world’s leading technology and information for life scientists.

Headquarters

850 Lincoln Centre Drive

Foster City, CA 94404 USA

Phone: +1 650.638.5800

Toll Free (In North America): +1 800.345.5224

Fax: +1 650.638.5884

06/2010

www.appliedbiosystems.com

Part Number 4365542 Rev. C

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