Bio-Rad iCycler iQ Instruction manual

iCycler iQ

™

Real-Time PCR Detection System

Instruction Manual

Catalog Number

170-8740

For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Safety Information

Important: Read this information carefully before using the iCycler iQ Real Time PCR Detection System.

Grounding

Always connect the iCycler Optical Module Power Supply to a 3-prong, grounded AC outlet using the AC power cord and external power supply provided with the iCycler iQ Real Time PCR Detection System. Do not use an adapter to a two-terminal outlet.

Servicing

The only user-serviceable parts of the iCycler are the lamp and filters. There are no other user-serviceable parts for this instrument. When replacing the lamp or filters, remove ONLY the outercasing of the iCycler Optical Module for lamp and filter replacement. Call

your local Bio-Rad office for all other service.

Power Switch

The external power supply must be placed so that there is free access to its power

switch.

Temperature

For normal operation the maximum ambient temperature should not exceed 30°C (see Appendix A for specifications).

There must be at least 4 inches clearance around the sides of the iCycler to adequately cool the system. Do not block the fan vents near the lamp, as this may lead to improper operation or cause physical damage to the iQ Detector.

Do not operate the iCycler Optical Module in extreme humidity (>90%) or where condensation can short internal electrical circuits or fog optical elements.

Notice

This Bio-Rad instrument is designed and certified to meet EN-61010 safety standards.

EN-61010 certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified in any way. Alteration of this instrument will:

• Void the manufacturer’s warranty.

• Void the EN-61010 safety certification.

• Create a potential safety hazard.

Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended, or by modifications of the instrument not performed by Bio-Rad or an authorized agent.

The iCycler is intended for laboratory research applications only.

Table of Contents

Page

Section 1 Introduction ..............................................................................................1

1.1 System Description ................................................................................................2

1.2 iCycler iQ Filter Instructions ..................................................................................4

Section 2 Quick Guide to Running an Experiment ................................................6

2.1 Introduction............................................................................................................6

2.2 Quick Guide to Single or Multi-Color Experimentation .........................................6

2.3 Well Factors ...........................................................................................................8

2.4 Pure Dye Calibration and RME Files .....................................................................9

Section 3 Introduction to the iCycler Program.....................................................11

3.1 Organization of the Program ................................................................................11

3.1.1 The Library Module ...............................................................................12

3.1.2 The Workshop Module...........................................................................12

3.1.3 The Run Time Central Module...............................................................12

3.1.4 The Data Analysis Module .....................................................................13

3.2 Organization of the Manual..................................................................................13

3.3 Definitions and Conventions ................................................................................13

3.4 Thermal Cycling Parameters ................................................................................13

3.4.1 Temperature and Dwell Time Ranges.....................................................14

3.4.2 Advanced Programming Options............................................................14

Section 4 The Library Module...............................................................................15

4.1 View Protocols Window ......................................................................................15

4.2 View Plate Setup Window....................................................................................16

4.3 View Quantities and Identifiers Window..............................................................18

4.4 View Post-Run Data Window ..............................................................................19

4.4.1 Opening Stored Amplification or Melt Curve Data Files........................19

4.4.2 Opening a Stored Pure Dye Calibration File...........................................19

4.4.3 Applying a New RME File to Stored Optical Data .................................20

Section 5 The Workshop Module...........................................................................21

5.1 Edit Protocol Window ..........................................................................................21

5.1.1 Quick Quide to Creating a New Protocol................................................22

5.1.2 Quick Quide to Editing a Stored Protocol...............................................24

5.1.3 Graphical Display...................................................................................26

5.1.4 Programming Dwell Times and Temperatures in the Spreadsheet..........26

5.1.5 Editing Cycles and Steps in the Spreadsheet...........................................27

5.1.6 Select Data Collection Step(s) Box.........................................................28

5.1.7 Programming Protocol Options in the Spreadsheet.................................28

5.1.8 Saving the Protocol.................................................................................34

5.2 Edit Plate Setup Window......................................................................................34

5.2.1 Quick Guide to Creating New Plate Setup in Whole Plate Mode............37

5.2.2 Quick Guide to Editing a Stored Plate Setup in Whole Plate Mode ........38

5.2.3 Quick Guide to Creating a New Plate Setup in Dye Layer Mode............39

5.2.4 Quick Guide to Editing a Stored Plate Setup in Dye Layer Mode...........40

5.2.5 Edit Plate Setup/Samples........................................................................41

5.2.6 Select and Load Fluorphores in Whole Plate Mode ................................48

5.2.7 Select Fluorophores in Dye Layer Mode ................................................49

5.3 View Quantities and Identifiers Window..............................................................50

5.4 Run Prep Window ................................................................................................50

5.4.1 Well Factors ...........................................................................................51

5.4.2 Pure Dye Calibration ..............................................................................55

Section 6 The Run Time Central Module .............................................................59

6.1 Thermal Cycler Window ......................................................................................59

6.1.1 Run-Time Protocol Editing.....................................................................60

6.1.2 Show Protocol Graph .............................................................................61

6.1.3 Show Plate Setup Grid............................................................................62

6.1.4 Pause/Stop ..............................................................................................62

6.2 Imaging Services..................................................................................................62

6.2.1 Description .............................................................................................63

6.2.2 Adjusting the Masks ...............................................................................64

6.2.3 Checking Mask Alignment.....................................................................65

6.2.4 Image File...............................................................................................65

Section 7 Data Analysis Module ............................................................................66

7.1 View/Save Data Window .....................................................................................66

7.1.1 View Plate ..............................................................................................67

7.1.2 Post-Run Editing ....................................................................................67

7.1.3 Saving OPD Files ...................................................................................68

7.2 PCR Quantification Window................................................................................71

7.2.1 Quick Guide to Collecting and Analyzing PCR Quantification Data......71

7.2.2 Amplification Plot ..................................................................................72

7.2.3 Amplification Plot Context Menu...........................................................72

7.2.4 PCR Quantification Data Display...........................................................79

7.2.5 Select Analysis Mode .............................................................................79

7.2.6 Select a Fluorophore...............................................................................80

7.2.7 Select Data Set........................................................................................80

7.2.8 Select Wells............................................................................................80

7.2.9 Threshold Cycle Calculation ..................................................................81

7.2.10 Save for x-axis/y-axis Allelic Analysis...................................................83

7.3 Standard Curve Window ......................................................................................83

7.4 Melt Curve Window.............................................................................................85

7.4.1 Quick Guide to Collecting and Analyzing Melt Curve Data ...................85

7.4.2 Melting Curve Plot .................................................................................85

7.4.3 Melting Curve Plot Context Menu..........................................................86

7.4.4 Melting Curve Data Spreadsheet ............................................................87

7.4.5 Select Wells............................................................................................88

7.4.6 Open/Save Settings.................................................................................88

7.4.7 Select Fluorophore..................................................................................88

7.4.8 Peak Bar .................................................................................................88

7.4.9 Temperature Bar.....................................................................................89

7.4.10 Edit Melt Peak Begin/End Temps...........................................................89

7.4.11 Delete Peaks...........................................................................................89

7.5 Allelic Discrimination Window............................................................................90

7.5.1 Quick Guide to Collecting and Analyzing Allelic Discrimination Data..90

7.5.2 Allelic Discrimination Plot .....................................................................92

7.5.3 Allelic Discrimination Plot Context Menu..............................................94

7.5.4 Allelic Data Spreadsheet.........................................................................94

7.5.5 Automatic/Manual Call ..........................................................................95

7.5.6 Display Mode .........................................................................................96

7.5.7 Vertical Threshold ..................................................................................96

7.5.8 Horizontal Threshold..............................................................................97

7.5.9 Normalize Data ......................................................................................97

Section 8 Care and Maintenance ...........................................................................98

8.1 Cleaning the Unit .................................................................................................98

8.2 Replacing the Lamp .............................................................................................98

Appendix A Specifications ..........................................................................................99

Appendix B Minimum Computer Specifications ....................................................100

Appendix C Warranty...............................................................................................101

Appendix D Product Information.............................................................................102

Appendix E Error Messages and Alerts...................................................................103

E.1 Software Startup.................................................................................................103

E.2 Workshop and Library........................................................................................103

E.3 Run Time Central...............................................................................................107

E.4 Data Analysis .....................................................................................................110

E.5 Exiting Software.................................................................................................111

Appendix F Hardware Error Messages ...................................................................112

Appendix G Description of iCycler and iQ Data Processing...................................114

Appendix H Uploading New Versions of Firmware.................................................120

Appendix I An example Pure Dye Calibration and RME file ...............................121

Appendix J Converting from Previous Versions of iCycler iQ Software ..............123

Appendix K Melt Curve Functionality.....................................................................124

Appendix L Filter Wheel Setup and Fluorophore Selection Guide........................136

Section 1 Introduction

The Polymerase Chain Reaction (PCR)* has been one of the most important developments in Molecular Biology. PCR has greatly accelerated the rate of genetic discovery, making critical techniques relatively easy and reproducible.

The availability of technology for kinetic, real time measurements of a PCR in process greatly expands the benefits of the PCR reaction. Real-Time analysis of PCR enables truly quantitative analysis of template concentration. Real-Time, on-line PCR monitoring also reduces contamination opportunities and speeds time to results because traditional post PCR steps are no longer necessary. A wide range of fluorescent chemistries may be employed to monitor the PCR in progress.

The iCycler Thermal Cycler provides the optimum performance for PCR and other thermal cycling techniques. Incorporating a Peltier driven heating and cooling design results in rapid heating and cooling performance. Rigorous testing of thermal block temperature accuracy, uniformity, consistency and heating/cooling rates insure reliable and reproducible experimental results.

The iCycler iQ Real Time PCR Detection System builds on the strengths of the iCycler thermal cycling system. The iCycler iQ system features a broad spectrum light source that offers maximum flexibility in selecting fluorescent chemistries. The filter based optical design allows selection of the optimal wavelengths of light for excitation and emission, resulting in excellent sensitivity and discrimination between multiple fluorophores. The 350,000 pixel array on the CCD detector allows for simultaneous imaging of all 96 wells every second. This results in a comprehensive data set illustrating the behavior of the data during each cycle. Simultaneous image collection insures that well-to-well data may reliably be compared. The iCycler iQ system reports data on the PCR in progress in Real Time, allowing immediate feedback on reaction success. All of these features of the iCycler iQ system hardware were built to promote reliability and flexibility.

The iCycler iQ Real Time Detection System Software includes the features that make software easy and useful. The software is designed for convenience–offering speedy setup and analytical results. The functions are presented graphically to minimize hunting through menus. Tips on usage are available as your mouse glides over the buttons - and the tips can be turned off when you no longer need to see them. The iCycler software automatically analyzes the collected data at the touch of a button yet leaves room for significant optimization of results based on your analysis preferences.

Fig. 1.1. Optical Module Upgrade to iCycler Thermal Cycler.

1.1 iCycler iQ System Description

The optical module houses the excitation system and the detection system. The Excitation system consists of a fan-cooled, 50-watt tungsten halogen lamp, a heat filter (infrared absorbing glass), a 6-position filter wheel fitted with optical filters and opaque filter “blanks”, and a dual mirror arrangement that allows simultaneous illumination of the entire sample plate. The excitation system is physically located on the right front corner of the optical module, with the lamp shining from right to left, perpendicular to the instrument axis. Light originates at the lamp, passes through the heat filter and a selected color filter, and is then reflected onto the 96 well plate in the thermal cycler by a set of mirrors. This light source excites the fluorescent molecules in the wells.

Registered User New User

er L

Fig. 1.2. Representation of Optical Detection System layout.

The detection system occupies the rear two-thirds of the optical module housing. The primary detection components include a 6-position emission filter wheel, an image intensifier, and a CCD detector. This filter wheel is identical to the wheel in the excitation system and is fitted with colored emission filters and opaque filter “blanks”. The intensifier increases the light intensity of the fluorescence without adding any electrical noise. The 350,000 pixel CCD allows very discrete quantitation of the fluorescence in the wells. Fluorescent light from the wells passes through the emission filter and intensifier and is then detected by the CCD.

Note: Suggested computer specifications for running the system software are given in Appendix B.

At the right side of the optical module are two connectors (see Figure 1.3):

• Round 9-pin power connector: This provides power to the optical module via

the optical system power supply. Note: Always turn power switch on the

power supply to the OFF position before connecting this connector.

• Parallel-port connector: This uses a cable that is 25 pin male-to-male and

connects to the computer. The computer requires an IEEE 1284 compatible,

8-bit bi-directional, or EPP type, parallel port. Data are transferred to the

computer via this cable.

At the right rear corner of the reaction module is a single connector.

• Miniature phone plug connector: This senses when the handle is lifted. When

the handle is lifted, the emission filter wheel shifts to the home position, blocking

light to the intensifier and the CCD detector.

At the left rear corner of the iCycler thermal cycler is a single connector.

• Serial connector: The iCycler program directs the operation of the iCycler via

this cable.

Intensifier

Emission

Filters

Excitation

Filters

Sample Plate

CCD Detector

Fig. 1.3. Side View of iCycler iQ Real-Time Detection System showing cable connections.

1.2 iCycler iQ Filter Description and Instructions

Before running the iCycler program, be sure the correct filters have been installed. In addition, if the system has been moved prior to use, it is necessary to check the alignment of the mask. This procedure is discussed in Section 6.2.2. All filters are mounted in holders (see Figure 1.4). The filter holders are held in filter wheels and may be changed. Each filter wheel holds six filters. Every position in a filter wheel must have a filter or an opaque filter blank to avoid damage to the CCD detector. The first position in each filter wheel is designated as the “home” position and must always contain an opaque filter blank.

Fig. 1.4. Filter in filter holder.

To change filters, proceed as follows:

1. Turn off the power to the Optical Module.

2. Release the two black latches on each side of the Optical Module. Slide the

housing backwards 2–3” (5–8 cm), exposing a black case, the filter wheel

housing. It is not necessary to remove the housing or cables.

Tab

TOP VIEW

Tab

Filter or Filter Blank

SIDE VIEW

3. To access the excitation filter slot, flip open the hatch on the right side of the

instrument. To access the emission filter, remove the plug from the slot located

on top of the instrument (see Figure 1.5). Changing both types of filters is

similar.

4. Turn the filter wheels to the desired positions using the supplied ball end hex

driver. As long as the power to the Optical Module is off, the filter wheels may

be turned freely in either direction.

5. To remove a filter, grasp it on both sides with the filter removal pliers and

squeeze the tab in; gently pull the filter up and out.

6. To insert a filter, grasp the filter with the pliers and insert it into a vacant slot. For

the excitation filters, the tab on the filter is toward the front of the instrument. For

the emission filters, the tab on the filter is toward the right of the instrument. Be

sure that every position in the filter wheel has either an excitation or emission

filter or a filter blank.

Record the position of filters to compare later with the plate setup. (See

Section 5.2)

7. After the filters or filter blanks have been inserted, replace the rubber plugs

over the slots of the filter wheels.

8. Move the camera housing forward and re-attach the latches.

Fig. 1.5. Installing the filters.

Section 2 The iCycler iQ Real Time PCR Detection System for Single and Multi-Color Experimentation

2.1 Introduction

The iCycler iQ Detector can simultaneously collect light from as many as four fluorophores in 96 wells and separate the signals into those of the individual fluorophores. This allows monitoring of up to four amplifications simultaneously on the same plate or in each well.

At each data collection step in real time, fluorescent light from each monitored well is measured through each filter pair. Since there is one filter pair for each fluorophore on the plate, each data collection step may require as many as four readings. For example, if there are probes labeled with FAM, HEX, Texas Red

and Cy™5 in a well, then at each data collection step, light from the well must be measured using the FAM filter pair, the HEX filter pair, the Texas Red filter pair and the Cy5 filter pair. The software then splits the signals into the contributions of each individual fluorophore. Using these data, separate amplification plots are displayed and at the end of the experiment, separate standard curves are automatically calculated for each fluorophore and unknown concentrations are determined on an individual fluorophore basis.

An experiment on the iCycler iQ system is defined by a Protocol file and a Plate Setup file. These files have the extensions .tmo and .pts, respectively. The output file of fluorescent data from which the amplficiation, melting curve, and standard curve data are extracted is called an Optical Data file and has the extension .opd.

In addition, every experiment on the iCycler iQ system requires well factor and pure dye calibration data in order to separate the signals of the individual fluorophores from the combined measured light. Well factors are generated for every individual experiment while pure dye calibration data persist from experiment to experiment. These two concepts are introduced below and presented in detail in Section 5.4. Understanding them will make it possible to rapidly optimize experimental protocol development and to collect the best possible optical data.

2.2 Quick Guide to Single or Multi-Color Experimentation

1. Allow the camera to warm up for 30 minutes. Power up the iCycler and log onto

the instrument. Open the iCycler software. If the iCycler or the iQ detector has

been moved since the last experiment, enter Imaging Services in the Run Time

Central module and check the alignment of the masks. See Section 6.2.

2. If necessary, conduct a Pure Dye Calibration protocol to collect the data

required to separate the signals from overlapping fluorophores. Calibration

data are required for each fluorophore/filter pair combination on the experimen-

tal plate. See Section 5.4.2.

Texas Red and SYBR are registered trademarks of Molecular Probes, Inc.

Cy is a trademark of Amersham Pharmacia Biotech.

3. Prepare the experimental PCR reactions in a 96-well Thin Wall plate (catalog

number 223-9441). Place a sheet of Optical Quality sealing tape (catalog number

223-9444) on the top of the 96-well plate. Use the tape applicator (flat plastic

wedge) to smooth the tape surface and tightly seal the tape to the plate. Avoid

touching the surface of the sealing tape with gloved fingers. Tear off the white

strips that remain on the sides of the tape. If individual sample tubes or strips of

tubes are to be used, you must seal the tubes with the appropriate caps. Note

that a minimum of 8 sample tubes is required to prevent tube crushing when

using the green anticondensation ring. If the ring is not present, a minimum of 14

sample tubes must be present.

4. Create and save the thermal protocol in the Workshop module. The thermal

protocol specifies the dwell times and set point temperatures, the number of

cycles, steps and repeats, and the step(s) at which data collection are to occur.

See Section 5.1.

5. Create and save the Plate Setup in the Workshop module. In the Plate Setup

window, indicate what fluorophores are to be monitored in which wells and

define the sample type, and for Standards, enter the quantity and units of

measure. Check these entries in the ‘View Plate Setup’ tab before proceeding.

See Section 4.2. The process of creating the Plate Setup includes choosing the

appropriate Filter Wheel Setup file. Choose a Filter Wheel Setup that includes

all the fluorophores that you want to monitor. See Appendix L.

6. Ensure that the positions of the filters in the excitation (lamp) and emission

(camera) filter wheels are in the exact same position as defined by the filter

wheel setup chosen in Step 5. See Appendix L.

7. If you will be using an external well factor plate (see Section 2.3), place the well

factor plate in the iCycler; otherwise, place the experimental plate in the

iCycler. Click the View Protocol tab in the Library module and select the

desired Thermal Protocol; click the View Plate Setup tab in the Library module

and select the desired plate setup and then click Run.

8. In the Run Prep tab, confirm that the desired protocol and plate setup files are

selected. Enter the reaction volume. Indicate the type of protocol (PCR

Quantification/Melt Curve or Pure Dye Calibration) and the Well Factor Source,

then click Begin Run. (Figure 2.1)

Fig. 2.1. Run prep tab.

9. Enter a name for the data file. Data are saved during the running protocol. The

run will not begin without a data filename.

10. Well factors from the Experimental Plate will be collected automatically after you

click Begin Run and the protocol will execute immediately afterwards without

any user intervention. If you are using a Well Factor Plate, the protocol will begin

execution and after about five minutes, the external well factors will be collected

and the iCycler will go into Pause mode. During the Pause, remove the well

factor plate and replace it with the experimental plate and then click Continue

Running Protocol. (See Figure 2.6)

11. After data collection on the PCR reaction plate begins, the PCR Quantification

plot will be displayed and the software will open the Data Analysis module. It is

not possible to make adjustments to the PCR Quantification plot while data are

being collected. You can change the monitored fluorophore or adjust the size

of the plot during steps at which data are not collected.

2.3 Well Factors

Well factors are used to compensate for any system or pipetting non-uniformity in order to optimize fluorescent data quality and analysis. Well factors must be collected at the beginning of each experiment. Well factors are calculated after cycling the filter wheels through all monitored positions while collecting light from a uniform plate. Well factors may be collected directly from an experimental plate or indirectly from an external source plate.

The better and easier source of well factors is the actual experimental plate. Well factors collected from the experimental plate are called dynamic well factors. The only requirement for using dynamic well factors is that each monitored well must contain the same composition of fluorophores. Within each dye layer the fluorophore must be present at the same concentration, however, all dye layers need not have the same concentration. If all the wells on a plate have, for example, 50 nM fluorescein, 100 nM HEX, 125 nM Texas Red and 200 nM Cy5, you can use dynamic well factors because the fluorophore composition is the same in every well. If some of the wells have 100 nM fluorescein and others have 200 nM fluorescein, then you cannot use dynamic well factors and you must use external well factors. Collection of dynamic well factors is a completely automated process initiated by clicking the Experimental Plate radio button in the Run Prep screen (see Figure 2.1).

In most experiments using DNA-binding dyes like SYBR®Green I or ethidium bromide, dynamic well factors cannot be used. When the template DNA is denatured, the fluorescence of the intercalators is not sufficiently high to calculate statistically valid well factors. There are three solutions to this problem: (1) use iQ SYBR Green Supermix (Catalog # 170-8880) which already includes flourescein (2) use external well factor plate or (2) for experiments with SYBR Green I, spike the master mix with a small volume of dilute fluorescein solution (see Section 2.3.2). This dilute fluorescein results in sufficient fluorescence at 95°C so that good dynamic well factors can be calculated and it will not interfere with the PCR.

The details of preparing and using well factor plates are in the section describing the Run Prep window of the Workshop module (Section 5.4).

Fig. 2.2. Well Factor guidelines.

2.4 Pure Dye Calibration and RME Files

Pure dye calibration data are required for every experiment, even those that monitor only one fluorophore, however, they need not be collected at each experiment. In contrast to well factors which must be collected at the beginning of each experiment, pure dye calibration data persist from experiment to experiment. Only when a new combination of fluorophore and filter pair are added to the analysis a new pure dye calibration must be done.

The pure dye calibration data are used to separate the total light signal into the contributions of the individual fluorophores. The data are collected by executing a Pure Dye Calibration protocol on a plate containing replicate wells filled with a single pure dye calibrator solution; there is a different calibrator solution for each fluorophore. These data are written to the file RME.ini found at C:\Program Files\Bio-Rad\iCycler\Ini. Pure dye calibration data are collected on as many as 16 combinations of fluorophore and filter pairs per protocol (up to four fluorophores and up to four filter pairs). At the end of the calibration protocol, the data are automatically written to the RME file: one entry for each fluorophore/filter pair combination. Once pure dye calibration data are written to the RME file they are valid on all subsequent experiments using the same fluorophore/filter pair combinations. As more pure dye calibration protocols are executed, the RME file is updated with data for the new fluorophore/filter pair combination. If a fluorophore/filter pair combination for which an RME entry exists is repeated in a subsequent pure dye calibration protocol, the new results are written to the RME file and the old results are overwritten.

EXPERIMENTAL

PLATE FACTORS

(Dynamic)

If a plate setup is specified that includes a fluorophore/filter pair combination for which pure dye calibration data do not exist, an alert message will be presented when the experiment is initiated. The RME file must be updated with the necessary pure dye calibration data before conducting the experiment. It is a good practice to archive RME files before running new pure dye calibration protocols. Archive the RME files by renaming them or moving them from the C:\Program Files\Bio-Rad\iCycler\Ini folder.

Every time an optical data file is created, the RME data present at the time of the experiment are saved into the file. This permits data files to be opened on other computers and also allows the original RME data to be maintained after subsequent recalibrations. It is also possible to overwrite the RME data that were used at the time of the optical data collection if the pure dye calibration data were suspect or corrupted.

There is an example of a pure dye calibration and RME file in Appendix I.

The details of preparing a pure dye calibration plate and performing the pure dye calibration are in the section describing the Run Prep window of the Workshop module (Section 5.4).

Section 3 Introduction to the iCycler Program

3.1 Organization of the Program

The iCycler program allows you to create and run thermal cycling programs on the iCycler and to collect and analyze fluorescent data captured by the Optical Module. The operation of each instrument is controlled by separate parts of the iCycler Program. Operation of the iCycler thermal cycler is controlled by a segment of the program called ‘Protocols’. Protocol files are thermal cycling programs that direct the operation of the iCycler. The Protocol files also specify when data will be collected during the thermal cycling run. Protocol files are stored with a ‘.tmo’ extension. The details of setting up protocol files are described in Section 5.1.

Fig. 3.1 Layout of a screen.

Operation of the Optical Module is controlled by the part of the program called ‘Plate Setup’. This portion of the program allows you to specify from which sample wells data will be collected, the type of sample in each well (e.g., standard, unknown, control, etc.) and the fluorophores to be monitored. Plate setup files are stored with a ‘.pts’ extension. The details of setting up Plate Setup files are described in Section 5.2. In order to run a thermal cycling program and collect fluorescent data both a Protocol file and a Plate Setup file must be specified.

The iCycler Program is organized into four sections, called ‘modules’. These are the Library Module, the Workshop Module, the Run Time Central Module and the Data Analysis Module. Icons representing each of the modules are always shown on the left side of the screen. The module that is opened is displayed with a highlighted border while the names of the other modules have plain borders. Figure 3.1 shows the first screen you see when you open the iCycler Program: the names of all of the modules are listed on the left side of the screen with the Library Module icon highlighted, and the Library Module window is displayed on the entire right side of the screen. Each module has a different function, described below.

3.1.1 The Library Module

The Library Module contains Protocol, Plate Setup, and Data files. In the Library you may:

• View Protocol files

• View Plate Setup files

• View Quantities and Identifiers for the Plate Setup file

• Select Data files to view in the Data Analysis Module

• Select Protocol files to edit in the Workshop Module

• Select Plate Setup files to edit in the Workshop Module

• Initiate a run using stored Protocol and Plate Setup Files

3.1.2 The Workshop Module

The Workshop Module allows you to work with Protocol and Plate Setup files. Use this module to:

• Edit existing Protocol files

• Edit existing Plate Setup files

• Create new Protocol files

• Create new Plate Setup files

• Save new and edited Protocol files

• Save new and edited Plate Setup files

• Initiate a run using new or edited Protocol and Plate Setup files.

Since Protocol and Plate Setup files are written and saved separately, you may “mix and match” a different file from each category in new experiments.

3.1.3 The Run Time Central Module

Once settings are confirmed in the Run Prep section of the Workshop Module, the iCycler Program transfers you directly into the Run Time Central Module where you may monitor the progress of the reaction, including the start and completion time for the experiment, the current cycle, step, and repeat number and the thermal activity of the iCycler thermal cycler.

3.1.4 The Data Analysis Module

This module may be accessed in either of two ways.

1. It opens automatically from the Run Time Central Module when the iCycler iQ

Real Time PCR Detection System begins collecting fluorescence data which

are being analyzed in real time. This allows you to monitor a reaction as it

occurs.

2. You may open a stored data set from the Library Module and the data will

automatically be presented in the Data Analysis Module.

This module allows you to:

• View experimental data

• Optimize data

• Assign threshold cycles for all standards and unknowns

• Construct standard curves

• Determine starting concentrations of unknowns

• Conduct statistical analyses.

3.2 Organization of the Manual

The four sections that follow this one describe each of the four modules. Within each section, the manual is organized by the separate windows of the module.

3.3 Definitions and Conventions

The following customs have been adopted in the text of this instruction manual:

• A “window” refers to the view of the iCycler Program found on the computer

screen.

• Active buttons across the top of a window are referred to as ‘tabs’.

• A text box refers to a field in the window that you can type in.

• A field box refers to a region in the window that you cannot type in but provides

information about the program.

• A dialog box refers to a region in the window that allows you to make a selection.

• All active buttons are printed in bold type in the text descriptions and figure

legends. For example, the Edit button is always printed in bold since selecting

this will result in some action by the iCycler Program.

3.4 Thermal Cycling Parameters

Protocol files contain the information necessary to direct the operation of the iCycler. A protocol is made up of as many as nine cycles, and a cycle is made up of as many as nine steps. A step is defined by specifying a setpoint temperature and the dwell time at that temperature. A cycle is defined by specifying the times and temperatures for all steps and the number of times the cycle is repeated. Cycles may be repeated up to 600 times.

3.4.1 Temperature and Dwell Time Ranges

Temperatures between 4.0 and 100.0 °C may be entered for any setpoint temperature.

Finite dwell times may be as long as 99 minutes and 59 seconds (99:59) or as short as 1 second (00:01).

Zero Dwell Times. When the dwell time is set to 00:00, the iCycler will heat or cool until it attains the setpoint temperature and then immediately begin heating or cooling to the next setpoint temperature.

3.4.2 Programming Options

Many advanced options are available for thermal protocols. They are listed below and detailed in the section describing the Edit Protocol window of the Workshop (Section 5.1).

• Infinite Hold. Holds a defined temperature indefinitely until user intervention.

• Gradient. Allows a reproducible gradient of between 1 and 25 C to be

programmed down-the-block during any single step.

• Melt Curve. Enables collection of data over a specified temperature range to

collect melting curve data for analysis.

• Increment/Decrement Temperature. Defines a periodic incremental increase

or decrease in temperature during a repeated cycle.

• Increment/Decrement Time. Defines a periodic incremental increase or

decrease in dwell time during a repeated cycle.

• Ramping. Allows specification of the rate of heating and cooling.

• Cycle Description. Names the cycles of the protocol.

• Step Process. Names the steps of a cycle.

Section 4 The Library Module

From the Library module you may initiate experiments using saved protocol and plate setup files, view saved Protocols and Plate Setup files and open saved Optical Data files. The Quantities and Identifiers of the currently selected plate setup file and any notations about the currently selected optical data file are also available in the Library. Protocol and plate setup files viewed in the Library may be selected for editing in the Workshop or you may choose to create a new protocol or plate setup in the Workshop.

The Library module consists of four windows; each is accessed by its associated tab.

• View Protocol: Allows navigation of stored protocol files. Provides information

on the thermal parameters for the protocol specified and indicates when data

will be collected and analyzed (see Figure 4.1).

• View Plate Setup: Allows navigation of stored plate setup files. Provides

information about the location of sample wells, the sample type and the

fluorophores that will be analyzed in each (see Figures 4.2 and 4.3).

• View Quantities and Identifiers: Displays information entered by the user

when the plate setup was created (see Figure 4.4).

• View Post-Run Data: Allows navigation of stored data files. Displays any

notes entered by user. Permits opening of stored data files (see Figure 4.5).

4.1 View Protocol Window

The View Protocol window of the Library module is the first window that appears when the iCycler program is opened (see Figure 4.1). In this window, you can navigate the directory of protocol files. When a protocol file name is highlighted in the directory tree, the details of the protocol are displayed in the View Protocol window.

The lower half of the window displays the protocol file identified in the Viewing Protocol field both graphically and in spreadsheet format (see Figure 4.1). The graphical display shows the reaction temperature (on the y-axis) as a function of time (on the x-axis). In the graphical display:

• The bar across the top of the graphical display shows the cycle number;

• The numbers below the bar indicate the setpoint temperature for each step in

the cycle (i.e., the y-axis on the graph) and the dwell time specified for that step

(i.e., the x-axis on the graph).

• The presence of a camera icon on a particular cycle of the graphical display

indicates that optical data will be collected at that step. A yellow camera icon

indicates that amplification data will be collected while a green camera icon

indicates collection of melt curve data.

Details of specialized options, such as automatic increment and decrement of temperature or dwell time are provided in the spreadsheet but not in the graphical display.

Fig. 4.1. Library / View Protocol Window. This is the default window that appears upon opening the

iCycler Program.

The upper part of the window displays the following protocol file information.

• The Drive Location: The Protocol files shown here are stored on the C drive.

• The Directory Tree: The Protocol files shown here are stored in the User1 folder.

• The Protocol Files menu: A list box of all protocol file names in the directory

identified in the Directory Tree. All protocol filenames have a .tmo extension.

• The Viewing Protocol field box: The file name of the protocol displayed in this

window.

• The Selected Plate Setup field box: The currently loaded plate setup file.

The right side of the window has the following active buttons.

• E

dit this protocol: Transfers the selected Protocol file to the Edit Protocol

window of the Workshop; this allows you to edit the protocol displayed on the

screen.

• Create a new protocol: Transfers to the Edit Protocol window of the

Workshop for creation of a new protocol file.

• Run with selected plate set up: Transfers the selected protocol file and the

selected plate setup file to the Run Prep window of the Workshop for initiation

of an experiment.

• Print this protocol: Prints the spreadsheet section of the selected Protocol.

4.2 View Plate Setup Window

In this window, you can navigate the directory of plate setup files. When a plate setup file name is highlighted in the directory tree, the details of the plate setup, including the monitored wells, the sample types loaded in each well and the fluorophores loaded in each well, are displayed in the View Plate Setup window (Figure 4.2).

Fig. 4.2. Library / View Plate Setup Window, Fluors and Samples, Show All Fluors View.

The upper part of the View Plate Setup window is similar to the upper part of the View Protocol window (compare Figures 4.1 and 4.2.)

The View Plate Setup window displays the following plate setup file information:

• The Drive Location: Plate setup files are stored on the C drive.

• The Directory Tree: The directory location of the current plate setup; plate

setup files are stored in the User1 folder.

• The Plate Setup Files menu: A list box of all plate setup file names in the

directory identified in the Directory Tree; all plate setup filenames have a .pts

extension.

• The Viewing Plate Setup field: The filename of the plate setup displayed in this

window

• The Selected Protocol field: The filename of the currently selected protocol file.

The View Plate Setup window has the following active buttons:

• Edit

this plate setup: Transfers the selected Plate Setup file to the Edit Plate Setup window of the Workshop; this allows you to edit the plate setup displayed on the screen.

• Create a new plate setup: Transfers to the Edit Plate Setup window of the Workshop; this allows you to create a new plate setup beginning with a blank plate layout.

• Run

with selected protocol: Transfers the selected plate setup file and the currently selected protocol file to the Run Prep window of the Workshop for initiation of an experiment.

There are two buttons above the sample plate grid.

• Fluors and Samples. A list of all fluorophores defined for the selected plate setup file is displayed in the box on the right. When one of these fluorophore names is highlighted, the corresponding sample type for each well containing that fluorophore is displayed on the grid. When Show All Fluors is selected, then the wells of the grid are filled with the corresponding color of each fluorophore monitored in that well. When Show All Samples is selected then the wells of the grid are filled with the appropriate sample type icons.

• Notes: Displays any notes written about the plate setup (see Figure 4.3).

Fig. 4.3. Library / View Plate Setup window, Notes view.

4.3 View Quantities and Identifiers Window

This window displays standard quantities and identifiers for every well of the

plate setup currently selected in the View Plate Setup Window. Data are displayed one dye layer at a time.

Fig. 4.4. Library / View Quantities/Identifiers window.

4.4 View Post-Run Data Window

The View Post-Run Data window is used to navigate the directory tree of opti-

cal data files. When an optical data file name is highlighted, the following information is displayed:

Fig. 4.5. Library / View Post-Run Data Window.

• The name of the optical data file is shown in the Selected Data File field box:.

• The protocol used.

• The plate setup used.

• Data Run Notes: These are notes entered by the user at the time of the run.

• The Filter Wheel Setup used in the experiment.

4.4.1 Opening Stored Amplification or Melt Curve Data Files

To open an optical data file from an amplification or melt curve experiment,

select its name in the directory tree and click Analyze Data.

4.4.2 Opening a Stored Pure Dye Calibration File

If the selected optical data file is a pure dye calibration file, then the

Calibration button will be active. Click this button to open the pure dye calibration optical data file. Note: opening a stored pure dye calibration optical data file

will cause the current RME file to be overwritten with the data in the selected pure dye calibration file. All subsequent experimental data will be collected and

analyzed with the new RME file. See the discussion on Pure Dye Calibration and RME files in Section 5.4 and Appendix I.

4.4.3 Applying a New RME File to Stored Optical Data

When an optical data file is saved, the RME values are written to the file along

with the collected experimental data making it possible to analyze the data on a computer without RME data or a computer with different RME data. Any time that the optical data file is subsequently opened with the iCycler program, the data are analyzed using the values that were in the RME.ini file when the optical data were collected. It is possible to overwrite the RME values stored within the OPD file with the ones in the current RME.ini file, thus changing the analysis of the experimental data. This feature protects you from losing valuable experimental data if a pure dye calibration was conducted incorrectly.

Fig. 4.6. Enabling external calibration file option.

In order to apply the current RME values to a stored data set, highlight the

name of the optical data file in the directory tree and then click the box labeled Use External Calibration File. The optical data file will be opened and the new RME values will be applied to the data. The existing RME values will be overwritten if you save the file again. If the software is exited without explicitly saving the data set again the original RME values will be restored. Optical data may be analyzed repeatedly with different RME files simply by exchanging the location of stored (inactive) RME files with the active RME file. The only active RME file is the one stored at C:\Program Files\Bio-Rad\iCycler\Ini.

Note: It is recommended that the optical data file is saved under a different name before saving the file with new RME values. This ensures that the original file is always maintained for reference.

Section 5 The Workshop Module

The Workshop module is where Protocol and Plate Setup files are edited

and created. Section 5.1 describes the layout of the Edit Protocol window and instructions for creating and editing protocol files. Section 5.2 describes the organization of the Edit Plate Setup window and explains how to create and edit plate setup files.

There are four windows in the Workshop:

• Edit Protocol: Protocol files are created, edited and saved in this window. You can initiate an experiment with the displayed protocol, once it is saved, and the currently loaded plate setup from this window.

• Edit Plate Setup. Plate setup files are created, edited and saved in this win- dow. You can initiate an experiment with the displayed plate setup, once it is saved, and the currently loaded protocol from this window.

• View Quantities/Identifiers: This window displays information entered in the Edit Plate setup window on an individual dye-layer basis.

• Run Prep: When you choose to run a protocol or plate setup from either the Workshop or the Library, this is window in which you confirm the protocol file, plate setup file, and conditions for the run. The experiment begins when you click Begin Run in this window.

5.1 Edit Protocol Window

New protocols are created and existing protocols are edited in the Edit Protocol

window. (Figure 5.1)

Fig. 5.1. Workshop / Edit Protocol Window. This shows the minimum programming spreadsheet.

A graphical display of the currently loaded protocol, showing reaction temperatures

(on the y-axis) and dwell times (on the x-axis), is shown in the middle one-third of the window. The actual thermal protocol is displayed in an adjustable spreadsheet at the bottom of the window.

This window contains:

• The Show options box lists advanced options that can be applied to the protocol (see below).

• The Protocol Filename text box contains the protocol filename.

• The Save this Protocol and Run with selected plate setup buttons are used to save changes to the protocol and to run the protocol, respectively (see Sections 5.1.8 and 5.4);

• The Select data collection step(s) box may be used to specify the step at which the data are collected. This is described below.

The procedures for creating new protocols and editing existing ones are

summarized in Sections 5.1.1 and 5.1.2. The graphical display is described in Section 5.1.3. Programming in the spreadsheet and specifying optical data collection are described in detail in Sections 5.1.4–5.1.7, and the saving of protocol files is detailed in Section 5.1.8.

5.1.1 Quick Guide to Creating a New Protocol

1. From the Library module, select the View Protocol tab.

2. Click Create a new protocol. The Edit Protocol window of the Workshop module will open.

3. Start in the spreadsheet at the bottom left of the window and fill in the thermal cycling protocol. As you make changes in the spreadsheet, they are reflected in the graphical representation at the top of the window. The cycle being edited is shown in blue on the bar across the top of the graph and is highlighted in blue in the spreadsheet.

• Double click in a time or temperature field to change the default settings.

• Insert a new cycle in front of the current cycle by clicking Insert Cycle and

then clicking anywhere on the current cycle in the spreadsheet. Insert a cycle after the last cycle by clicking Insert Cycle and then clicking anywhere on the first blank line at the bottom of the spreadsheet. If you right mouse click on the Insert Cycle button, you can choose to insert a 1-, 2- or 3-step cycle. When finished, deselect Insert Cycle.

• Delete cycles by clicking Delete Cycle and then clicking anywhere on that

cycle in the spreadsheet. When finished, deselect Delete Cycle.

• Insert a step in front of the current step by clicking Insert Step and then clicking

on the current step. If you right mouse click on the Insert Step button, you can choose to insert the new step before or after the current step. When finished, deselect Insert Step.

• Delete steps by clicking Delete Step and then clicking anywhere on the

line containing the step to be deleted. When finished, deselect Delete

Step.

4. If you want to add any protocol options, first enable them by clicking in the check box next to its description in the Show options box.

• Infinite Hold. A column labeled Hold will appear in the spreadsheet. Click

the Hold button on any step of a non-repeated cycle and the set point temperature will be maintained indefinitely until user intervention. A red check mark will appear in the Hold column of the spreadsheet. Turn off the Hold by clicking on the red check mark. The software will not allow you to program a Hold if the cycle is repeated.

• Gradient. Two new columns appear in the spreadsheet. Click the Gradient

button on the desired step. Specify the range of the gradient in the other column. The range may be from 1° to 25°C across the block and the block temperatures must be in the range of 40° to 95°C.

• Melt Curve. Three new columns appear in the spreadsheet. Click the Melt

Curve column on the step at which melt curve data are to be collected. Indicate the temperature increment or decrement with each cycle. A green camera will appear on the corresponding step in the Select Data Collection Step box.

• Increment Temperature /Decrement Temperature. Three new columns will

appear. Specify the increment or decrement in temperature, the first cycle at which the change is to occur and the frequency with which the change is to occur. For example, to increase temperature by 0.2°C beginning with the third cycle and to further increase the temperature every other cycle after that you would enter 0.2 in the + Temp column, 3 in the Begin Repeat column and 2 in the How Often column.

• Increment Time /Decrement Time. Three new columns will appear.

Specify the increment or decrement in time, the first cycle at which the change is to occur and the frequency with which the change is to occur. For example, to decrease time by 2 seconds beginning with the third cycle and to further decrease the time every other cycle after that, you would enter 00:02 in the - Time column, 3 in the Begin Repeat column and 2 in the How Often column.

• Ramping. Double click in the Ramping column and choose MIN or MAX

or make a direct entry of heating or cooling rate. Valid entries are from

0.1° to 3.3°C/sec for heating steps and 0.1° to 2.0°C/sec for cooling steps.

• Cycle Description/Step Process. One new column appears if either of

these is selected. Choose a descriptive name from the pull down menu or enter one directly.

5. In the Select data collection step(s) box, specify the cycles at which fluores- cent data are to be collected for quantitative experiments.

• Click once in a camera square to indicate that data are to be collected for

post-run analysis. A camera with a yellow lens will appear in the box and on the graphical display.

• Click a second time to indicate that fluorescent data are to be collected

and analyzed in real time. REAL TIME will appear next to the yellow camera in the Select data collection step(s) box.

• Click a third time to deselect data collection for that cycle. The camera

icon will disappear.

6. Double click in the Protocol File Name text box and enter a new name for the protocol.

7. Click Save this protocol. A Save dialog box will appear, click Save again.

Note: You may save plate setup and protocol files to the iCycler folder or any subfolder of the iCycler folder.

5.1.2 Quick Guide to Editing a Stored Protocol

1. From the Library module, select the View Protocol tab.

2. In the top left corner of the window, select the drive where the stored plate setup file resides.

3. Use the directory tree to locate the folder containing the stored protocol file.

4. Select the desired file from the Protocol Files box.

5. Click Edit this protocol. The Edit Protocol window of the Workshop module will open.

6. Start in the spreadsheet at the bottom left of the window and fill in the thermal cycling protocol. As you make changes in the spreadsheet, they are reflected in the graphical representation at the top of the window. The current cycle begin edited is shown in blue on the bar across the top of the graph and is highlighted in blue in the spreadsheet.

• Double click in a time or temperature field to change the settings.

• Insert a new cycle in front of the current cycle by clicking Insert Cycle and

then clicking anywhere on the current cycle in the spreadsheet. Insert a cycle after the last cycle by clicking Insert Cycle and then clicking anywhere on the first blank line at the bottom of the spreadsheet. If you right mouse click on the Insert Cycle button, you can choose to insert a One-, Two- or three-step cycle. When finished, deselect Insert Cycle.

• Delete cycles by clicking Delete Cycle and then clicking anywhere on that

cycle in the spreadsheet. When finished, deselect Delete Cycle.

• Insert a step in front of the current step by clicking Insert Step and then

clicking on the current step. If you right mouse click on the Insert Step button, you can choose to insert the new step after the current step. When finished, deselect Insert Step.

• Delete steps by clicking Delete Step and then clicking anywhere on the

line containing the step to be deleted. When finished, deselect Delete Step.

7. If you want to add any protocol options, choose them by clicking in the check box next to its description in the Show options box.

• Infinite Hold. A column labeled Hold will appear in the spreadsheet. Click the

Hold button on any step of a non-repeated cycle and the set point temperature will be maintained indefinitely until user intervention. A red check mark will appear in the Hold column of the spreadsheet. Turn off the Hold by clicking on the red check mark. The software will not allow you to program a Hold if the cycle is repeated.

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Bio-Rad iCycler iQ Instruction manual

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