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Techne PrimeQ Operator's Manual

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PrimeQ
OPERATOR'S MANUAL
Version 2.1
08/12

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  • Page 1 PrimeQ OPERATOR’S MANUAL Version 2.1 08/12...
  • Page 3 Veuillez lire attentivement toutes les instructions de ce document avant d’utiliser l’appareil. This operator guide provides the basic information to get you started on the PrimeQ real-time PCR system. It covers everything from taking the instrument out of the box, installing the application software and system set-up, to setting up, running and analysing a range of real-time PCR experiments.
  • Page 4 Chapter 6 Troubleshooting and Identifying problems with the instrument and/or the Glossary Glossary of terms used in real-time PCR...

  • Page 7: Table Of Contents

    1.2.4 PrimeQ reports ......................16 1.2.5 Data Analysis ......................16 Unpacking ........................... 17 Warning! ..........................17 Uses of PrimeQ ........................17 Plates ..........................17 Thermal seal ........................18 Contacting us........................18 Installing the hardware ....................... 18 1.9.1 Site requirements ....................... 19 1.9.2...
  • Page 8 Inserting a previously run plate .................. 38 1.19.2 Running a plate ......................38 1.20 After use ......................... 39 Getting started .......................... 41 Introduction to PrimeQ ....................... 43 2.1.1 Principle of a real-time PCR instrument ..............43 2.1.2 Principle of PrimeQ ....................43 2.1.3 Applications of PrimeQ ....................
  • Page 9 3.2.2 The Editors ........................ 55 Using Quansoft ........................57 3.3.1 Home page ........................ 57 3.3.2 Main screen ....................... 58 3.3.3 Navigation bar ......................58 3.3.4 Title bar functions ...................... 59 3.3.5 Menu bar functions ....................60 3.3.6 Accessing the editors ....................61 Setting up an experiment ....................
  • Page 10 4.3.1 Viewing the results ....................115 4.3.2 PrimeQ Report ......................116 Passive reference dye (PRD) correction ................117 Analysis method: Baseline correction ................118 4.5.1 Assay requirements ....................118 4.5.2 Setup........................118 4.5.3 Viewing the results ....................120 4.5.4 PrimeQ Report ......................122 4.5.5...
  • Page 11 5.3.3 Replacing the fuses ....................175 Consumables........................175 Minimum computer requirements ..................176 Accessories ........................176 Replacement parts ......................177 Packing the PrimeQ instrument ..................178 5.8.1 Remove the filter cartridges ..................178 5.8.2 Remove the block ....................178 5.8.3 Packing the instrument ....................

  • Page 13: Introduction

    1 Introduction Safety and installation information About this chapter This chapter provides information on general safety aspects, definitions, advice and instructions for unpacking and installing your instrument. It also gives general information about the instrument and the control software, system requirements, basic procedures, control mechanisms and software installation.

  • Page 15: Primeq

    PrimeQ PrimeQ - the real-time nucleic acid detection system from Techne - has been designed with the advantage of an open chemistry format that allows the end user full flexibility in the methods and research they wish to pursue. This is complimented by the user-friendly application software, Quansoft, for easy setup of experiments and rapid analysis of data.

  • Page 16: Program Editor

    1.2.5.4 Allelic discrimination Users of PrimeQ have the option of this powerful technique capable of detecting single nucleotide differences (SNPs). It can be used to discriminate between genotypes, mutations and polymorphisms within or between samples simply by comparing the fluorescence signal obtained...

  • Page 17: Unpacking

    • Decontamination certificate • USB cable Keep the original packaging in case you ever need to return the unit for service or repair. Techne accepts no responsibility for damage incurred during transportation unless the unit is correctly packed and transported in its original packaging.

  • Page 18: Thermal Seal

    Always be certain that you use the film in the correct way. If you find that you have used the seal the wrong way up DO NOT PLACE IT IN THE PRIMEQ UNIT as it will damage the heated lid and this will invalidate the warranty.

  • Page 19: Site Requirements

    1.9.1 Site requirements PrimeQ can operate on a laboratory bench top. The unit is 420mm wide x 518mm deep x 528mm high and requires 100mm side clearance for operation. The unit weighs 25kg (55lb). The unit should ideally be operated in the temperature range of 18° C to 30° C out of direct sunlight and draughts.

  • Page 20: Operator Safety

    à l’appareil. 1.9.4 Installation 1. All Techne units are supplied with a mains cable. This is a plug-in lead type cable. 2. Before connecting the power supply, check the voltage against the rating plate. Connect the mains cable to a suitable supply according to the table below.
  • Page 21 I: power switch On 1.9.4.1 Instalación 1. Todos los aparatos Techne se suministran con un cable de alimentación. Puede ser fijo o independiente del aparato. 2. Antes de conectarlo, compruebe que el voltaje corresponde al de la placa indicadora. Conecte el cable de alimentación a un enchufe adecuado según la tabla expuesta a continuación.
  • Page 22 Le fusible (10A) à l’intérieur de l’appareil est destiné à assurer la protection de l’appareil et de l’opérateur. Les appareils fonctionner sur 100V et 230V La plaque d’identification se trouve à l’arrière de l’appareil. 3. Raccordez le câble d’alimentation à la prise située à l’arrière de l’appareil. 4.

  • Page 23: Before Switching On

    1.10 Before switching on 1.10.1 PrimeQ front view 1.10.2 Insert the block Push firmly on the front of the drawer to open it and insert the block. • Slide the quick release handles in the direction shown on the top to the unlock position.

  • Page 24: Connections On The Back Of The Unit

    D. Power connection. Ensure that the switch is in the off, O, position. Plug the mains cable into the power socket. When PrimeQ is sited in the correct position and all the connections have been made, turn the switch to the on (I)

  • Page 25: Technical Specification

    1.11 Technical Specification Thermal cycler Block Format: 96 x 0.2ml well low-profile micro tube plate Block Specification: 8 x Peltier block employing quad circuit technology to enhance performance Block Uniformity at 50ºC: < ± 0.25ºC Maximum Heating Ramp Rate: 2.2ºC/sec Temperature Range: 4ºC to 98ºC Sample Volume:...

  • Page 26: Working Conditions

    Please read this important GUARANTEE information. Notwithstanding the description and specification(s) of the units contained in the Operator Guide, Techne hereby reserves the right to make such changes as it sees fit to the units or to any component of the units.
  • Page 27 Please register online or complete and return the Registration Card to the address on the card. Returning the card or registering will help us to contact you with new information about Techne products and product up-dates thus improving our service to you.

  • Page 28: Installing The Application Software

    1.14 Installing the application software Always set your PC or laptop from which you are running PrimeQ so that standby is switched OFF. This can easily be achieved through the control panel or: • Right click on the desktop and select Properties.

  • Page 29: Loading Quansoft Onto The Pc

    Techne, you will be eligible for technical support and software updates posted on the Techne website. However, the software can still be used for a 28-day trial period without registration – simply click Register Later and the prompt screen will appear next time the software is opened.

  • Page 30: Software Upgrades

    3. Phone: Provide us with your details and registration number by telephoning: +44 (0)1785 810433. 1.14.4 Software upgrades Development of the PrimeQ control and analysis software is an ongoing process. To check your version number, click ‘Help/About Quansoft’ and a splash screen displaying the current details will appear. Check the Techne websites at www.techne.com...

  • Page 31: Using The Lcd Control Panel

    1.15.1 Function keys PrimeQ is controlled primarily from the PC using the application software, although some basic functions can be performed via the LCD screen located on the front of the instrument. The display indicates the instrument name, current status and basic information about any experiment currently running such as its progression.

  • Page 32: Before You Start

    3. The Quansoft software has been installed on the PC. 1.15.5 Start-up procedure 1. Turn on the power to PrimeQ by pressing the switch at the back of the instrument. A welcome beep can be heard. 2. Turn on the PC and wait for Windows to boot up.

  • Page 33: Filter Cartridge Care Instructions

    1.16.2 Filter cartridge care instructions • The filter cartridges must be handled with special care. • Only lift them from the foam in the box by the sides of the cartridges. • Avoid touching the filters. Loose particles should be removed with a bulb puffer or filtered, pressurized air cleaner. necessary, gently wipe the surface using 100% alcohol and optical wipes;...

  • Page 34: Adding A Filter Cartridge

    FAM or Cy5. The input table can hold details for the four filter positions of the carousel. • Click Next. A screen appears prompting the user to perform a five-step procedure. 1. Lift the top lid of the PrimeQ. 2. Lift the cartridge/carousel cover. 3. Insert the cartridge and ensure that the magnets engage to hold it in place;...

  • Page 35: Assigning Filter Descriptions In Quansoft

    1.16.5 Assigning filter descriptions in Quansoft Once the cartridge has been fitted into the cartridge carousel, the system will check for the presence of a new cartridge and then ask the user for the Dye Names. Up to four names can be assigned to any given filter cartridge and the colour coding can be changed by clicking on the Colour icon.

  • Page 36: Points To Remember

    1.17 Changing the instrument name All PrimeQ units are shipped with the name “PrimeQ” stored in the instrument’s software. This can be changed to a more appropriate name as required. • To change the instrument name, click on the Maintenance icon on the navigation bar. Then click on the Instruments icon.
  • Page 37 • On the instrument screen, click on Change, type in the new name and then click OK. Once the name has been changed you need to switch off the instrument before the new name can appear on the LCD screen and take effect. The maintenance screen also gives instrument-specific details including serial number, unique ID and block cycle count.

  • Page 38: Led Power Settings

    1.18 LED power settings The LED light source has variable power settings. This prevents PMT blinding and brings fluorescence into its linear range for certain applications. The role of this option is to cut down excess light from the LED source in high fluorescence applications. The three power options are: Low (50mA), Medium (100mA), and High (200mA).

  • Page 39: After Use

    1.20 After use When the samples have finished thermal cycling, remember that parts of the unit, such as the tubes, blocks and associated accessories, may be very hot. Take the precautions listed earlier. Después de su uso Cuando haya finalizado el calentamiento de muestras, recuerde que las piezas del equipo, tales como tubos, bloques y demás accesorios, pueden estar muy calientes.

  • Page 41: Getting Started

    Introduction to PrimeQ and real-time PCR About this chapter This chapter gives a description of PrimeQ together with a basic introduction to real-time PCR. It also provides examples of some of the fluorescent chemistries that can be used with PrimeQ.

  • Page 43: Introduction To Primeq

    The time taken for PrimeQ to read a plate can be adjusted by varying the integration time. Read times can be programmed from 250ms/well integration time (30 seconds/ 96-well plate) down to just 50ms/well (10 seconds/plate) for brighter fluorophores.

  • Page 44: Introduction To Real-Time Pcr

    Introduction to Real-time PCR 2.2.1 PCR PCR is a powerful biochemical technique that has revolutionised biological research by allowing minute amounts of DNA to be amplified millions of times in just a few hours. PCR allows the selective amplification of a ‘target’ region of DNA lying between two specific DNA sequences (primers).

  • Page 45: Real-Time Pcr On Primeq

    PCR product by using PrimeQ as a plate reader. In both approaches, the amount of product present in each reaction tube is visualized using fluorescent reporter dyes.

  • Page 46: Intercalating Dyes

    dye is excited at the appropriate wavelength should be proportional to the quantity of target DNA present. For each type of fluorescent chemistry, the fluorophore will emit a fluorescent light that is characteristic of the fluorophore used. A range of spectrally distinct fluorophores are commercially available, introducing the possibility of quantifying multiple targets with different probes in the same reaction well, otherwise known as multiplexing.

  • Page 47: Molecular Beacons

    fluorescence signal or fluorescence at a longer wavelength which is not detected. The 5’ fluorophore is often called the reporter. Mode of action of hydrolysis probes: During the PCR, as the DNA polymerase extends the upstream (forward) primer, it encounters the bound probe.

  • Page 48: System Overview

    System overview PrimeQ is a real-time PCR unit into which the operator places a 96-well plate containing the samples to be analysed. The unit is connected to a PC via the USB port on which the application software (Quansoft) is run. The software allows the user to: 1.

  • Page 49: The Optical Light Path

    To detector To detector To allow the PrimeQ instrument to be optimized for the use of specific fluorophore combinations, the user can choose to adjust the light intensity of the LED light source (50, 100 or 200mA settings). This is useful for obtaining balanced results when using both bright and weak chemistries/dyes in the same experiment.

  • Page 50: Scanning Module

    PrimeQ’s thermal cycler block. accommodates a low- profile 96-well plate. Each block is internally calibrated so that if the block replaced, temperature performance unchanged without having recalibrate instrument The thermal block fits into and can be accessed by opening the PrimeQ sample drawer. This provides...

  • Page 51: Quansoft

    3 Quansoft Using Quansoft About this chapter Accompanying PrimeQ is the intuitive, wizard-based software, Quansoft. This chapter discusses the concepts behind this software and looks at its application in the setting up and running of a real- time PCR experiment.

  • Page 53: Introducing Quansoft

    Introducing Quansoft PrimeQ is a real-time-PCR unit containing sophisticated thermal cycling and fluorescence detection technology. The user interacts with the system via Quansoft, the user-friendly Windows- based application software installed on a PC connected to the unit. The intuitive software allows the user to perform three main functions: 1.

  • Page 54: Software Overview

    Plate Layout Editor Within a matter of seconds, a 96-well microplate can be assigned with various sample types, including blanks, controls, standards or user-defined samples, all of which colour-coded ease identification. Results Editor Results for each stage of the program containing fluorescent readings are shown on individual tabs and can be analysed separately using various analysis methods.

  • Page 55: Home Page

    PrimeQ. Both the Plate Layout and Program Editors can be accessed from the Experiment Editor, and when combined with a set of analysis parameters, will produce a report of the results once the data has been collected.
  • Page 56 PCR run. The minimum requirement in order to start a run on PrimeQ is for the experiment to have a valid thermal cycling program – other settings such as plate layout and analysis method can be defined and/or changed post-run. These files can be saved to the Program Library in a .qprg format.

  • Page 57: Using Quansoft

    Quansoft’s Results Editor displays the results of the run, which can be sent to report or re-analysed with different parameters. Using Quansoft 3.3.1 Home page The Quansoft Home page acts as a gateway to the software functions, providing quick and easy links to both the Editors and their associated libraries.

  • Page 58: Main Screen

    3.3.2 Main screen Shortcuts for experiment setup and data analysis: Run an experiment: goes to the Experiment folder to select an experiment. Create a new experiment: goes to the Experiment Editor from where the user can create a new experiment or edit an existing one. Create a new program: goes to the Program Editor from where the user can create a new program or edit an existing one.

  • Page 59: Title Bar Functions

    The default supervisor password is techne - we suggest that you change this as soon as possible. Please ensure that you keep a record of the password as without it you will not be able to access these functions.

  • Page 60: Menu Bar Functions

    The Up button allows the user to navigate back to a previous folder (used if the directory structure has been turned off). The View button will change the way the files are displayed in the file view pane (detail, large/small icon, list views). The following buttons are displayed on the Title bar when using one of Quansoft’s Editors: The Finish button (Results Editor) saves the changes made and closes the editor.

  • Page 61: Accessing The Editors

    Help: Off- and on-line resources. Click Quansoft Help (short cut key F1) for access to this Operator guide, or click on Techne on the Web to link to the Techne website. Clicking on About Quansoft will bring up details about the software including the version and serial number.

  • Page 62: Setting Up An Experiment

    Setting up an experiment 3.4.1 An Overview Schematic diagram showing the options available for setting up an experiment on PrimeQ. The icons in the blue boxes represent the shortcut buttons available from the Home page. START 1: Choose to set up an entirely new experiment in the Experiment Editor, browse or edit previously saved plate layout or program templates or else access the Plate Layout and Program Editors direct from the Home page.

  • Page 63: Creating A New Experiment

    3.4.2 Creating a new experiment • From the Home page click on Create a New Experiment. A blank experiment template will open in the Experiment Editor. The Experiment Editor allows the user to perform three important functions: • Define a program, •...

  • Page 64: Setting Up A Program

    2. Plate Layout Setup: a. From New b. Browse an existing template. c. Edit the selected template 3. Analysis Method Selection This chapter will look at each of these functions in turn. 3.4.3 Setting up a program The PCR program is defined using the top-left area of the Experiment Editor main screen: There are three ways to define a PCR program: Click this button when the program is to be defined entirely from new.
  • Page 65 • Run information: The user can enter a user name and add any other comments relevant to the program. • Instrument settings: User-defined settings for heated lid temperature, disabling of the instrument buttons, plate type, sample volume and whether to read entire plate or include a final hold in the PCR program.
  • Page 66 • Heated Lid: Default 105° C. Set a temperature (possible temperature range from 100° C to 115°C). • Wait For…: Set a ‘wait for’ time (i.e. the time to wait while the lid heats up before the block thermal program begins, hrs:min:sec). Or click the button to wait until heated, the program will then start as soon as the lid reaches the set temperature.
  • Page 67 The default name New Stage can be changed if required. When a stage name is highlighted in the program file view, the stage parameters box in the centre of the screen allows the general parameters for that stage to be defined. •...
  • Page 68 • Temperature: Set using the sliding thermometer or type a value in the box. • Hold Time: Shown in hrs:min:sec up to a maximum of 99:59:59 (minimum 00:00:01). The default setting is seconds, therefore for a time in seconds, type in the number of seconds and press the enter key.
  • Page 69 change between these options. If the user is creating a new protocol and the instrument is not connected, it is not possible to select filter cartridges (the drop-down box is greyed-out). The user will be prompted to choose the filters on connection to the instrument or when trying to run the program.
  • Page 70 ® intercalating dye such as SYBR Green I is used, for confirmation that the correct product has been amplified. • Click Add Ramp to open the ramp read parameters box: • Start Temperature: Starting temperature of the ramp can be anywhere from 4°C. •...
  • Page 71 The read appears as a separate tab labelled according to the dye name. The temperature profile plot shows the reads as colour-coded points. Once the program is complete, clicking on the program name at the top level in the program file view will show the complete thermal plot: Any parts of the program, such as temperatures, hold times, read points, filters etc.
  • Page 72 • Highlight a folder and click Open to open up the program file in Experiment Editor. The template is ready to use. 3.4.3.8 Edit a program A program already present in the Experiment Editor can be edited simply and easily by clicking the Edit button in the Program pane of the Experiment Editor.
  • Page 73 • On the menu bar in Program Editor, select File and then Save As… • Change the file name if required. The file (.qprg format) will automatically be saved to the directory My Documents\Quansoft\Programs. Change the destination by browsing the file directories shown in the Save in: drop-down menu.

  • Page 74: Setting Up A Plate Layout

    3.4.4 Setting up a plate layout The role of the Plate Layout Editor is to define what types of samples go in to which wells of the plate. It is represented in the top-right panel of the Experiment Editor. The plate layout can be cleared or changed before or after the run.
  • Page 75 • Show all wells: The well information table will show only the details for the currently selected sample type, e.g. standards, unless this button is clicked. Click again to change back. • Reset names: Allows any user-defined changes to the names to be returned to the default. •...
  • Page 76 • Calibrator (CAL): In relative quantification methods, the ratio of the DNA template in different samples may be normalized to a calibrator sample. Particularly useful when comparing Cq values (section 4.7). • Reference Gene (REF): A common gene that is expressed in all samples at the same level. Can be useful in relative quantification methods.
  • Page 77 The custom sample type will now appear in the available list. Move over to the active list using the arrow if the sample type is to be used in the current experiment. Close the sample types setting box and return to the Plate Layout main screen. 3.4.4.5 Replicates Choose how many replicates there are for each sample from the scroll-down menu.
  • Page 78 The function buttons provide various tools that can assist in assigning the plate layout by simply clicking on the icon with the mouse so that it becomes highlighted. Clicking again de-activates the function. Erase: Clears individual wells that have been assigned a sample type. Fill by row: Allows quick-filling of the plate row by row.
  • Page 79 Choose user-defined names, concentrations and units of standards or any comments specific to a sample. The Well Information table will contain a complete list of all the sample types in the plate, but to list the details of just a selected sample type, click the Selected Sample Type button and then click on a sample type icon to choose which sample information to display.
  • Page 80 • To import from Excel into the Plate layout, select the cells to be copied and press Ctrl+C or select Copy. Note that the headings (consisting of Well, Type, Name, Concentration and Comment) also need to be copied for the data to considered valid while importing. •...
  • Page 81 3.4.4.9 Browse for an existing plate layout The user can browse for an existing plate layout file by clicking on the Browse button in the Plate layout pane of the Experiment Editor. This will open up the Plate Layout library folder and display any existing templates (.qpla files). •...
  • Page 82 IMPORTANT: Certain characters must not be used in the file name otherwise it may become corrupted and you may not be able to open the Results file. These characters are: < > & ‘ “ • Change the file name as required. If an existing name is selected then a warning message will appear asking if the file should be over-written.

  • Page 83: Defining The Analysis Method

    3.4.5 Defining the analysis method 3.4.5.1 Selecting an analysis method The Analysis Selection box in the Experiment Editor (also found in the Results Editor after the run has finished) allows the user to define the method of analysis to be applied to readings gathered during the PCR program.
  • Page 84 These will be dependent on the number of reads and the number of cycles programmed into the run (see Choosing an analysis method in section 4.2) PrimeQ supports the following analysis methods: • No analysis method defined (None) • Baseline correction •...
  • Page 85 • Dissociation curve • Plus/minus scoring • Allelic discrimination • Multi-read See Chapter 4 for a specific discussion of each analysis method in terms of requirements, setup and what each approach can reveal about the experimental data. 3.4.5.3 Assigning a dye usage As there may be more than one dye reading in a single stage, the role of each dye needs to be assigned.
  • Page 86 The readings are normalized by dividing the fluorescence of the reporter in each well by that of the PRD. This correction method is applicable to all the analysis types available on PrimeQ (see section 4.4 for more details). • Click Next.

  • Page 87: Saving An Experiment To The Library

    3.4.6 Saving an experiment to the library The experiment is now complete with a program file, plate layout and analysis method all defined. While the program and plate layout can be saved as separate files (shown in 3.4.3.10 and 3.4.4.12), the experiment can also be saved as a consolidated .qexp file at any point during the setup in the Experiment Editor.
  • Page 88 • Click Edit in the program or plate layout pane of the Experiment Editor and the appropriate editor will open. • Change settings in any step/stage, alter readings and so forth or delete the current template and import another from the library files. •...

  • Page 89: Running An Experiment

    3.5.1.1 Preparing the system • Turn on the power to PrimeQ using the power switch at the rear of the instrument. An idle screen will appear on the instrument’s LCD display. • Turn on the PC and log in under a user name.
  • Page 90 3.5.1.3 Running an experiment 3.5.1.4 Run to report Unless otherwise informed, the system will wait for an acknowledgment from the user that the run has ended before proceeding on to analysis and report generation. If Quansoft is required to run straight through to report generation with no prompt or user intervention then select the Run to Report option found under Tools on the Experiment Editor menu bar.
  • Page 91 • Save to the Experiment library folder if required (see section 3.4.6). Note that saving will over-write the previous version of the experiment. IMPORTANT: Certain characters must not be used in the file name otherwise it may become corrupted and you may not be able to open the Results file. These characters are: <...

  • Page 92: Monitoring The Run

    3.5.1.7 Additional information If the instrument was not connected when the experiment was created, the correct cartridges will need to be selected for each read. To select the cartridges, go into the Program Editor from the Experiment Editor and assign cartridges to each read. Alternately when the run button is pressed the following error window will appear: Selecting OK will take the user to the correct window where all of the reads can be assigned a filter cartridge.
  • Page 93 3.5.2.2 From the Run Screen As the PCR is in progress, the Run Screen displays the current status of the program in real time. Fluorescence data is shown on a per-well basis with the different stages of the run (which have readings) being displayed as separate tabs.

  • Page 94: Stopping Or Pausing A Run

    • Fluorescence graph: Data for a particular stage will be displayed in the graph. Choose to display readings for the entire plate or just for selected wells. Change the dye being viewed using the Dye to View option. • Well view: A fluorescence curve is displayed in each well as the PCR progresses. Highlight a well(s) with the mouse to display the fluorescence data for that well(s) in the adjacent graph.

  • Page 95: Led Intensity Settings

    LED intensity settings Please note the following: • If the fluorescent counts on the raw data plot of the Run Screen or Results Editor are above 100,000 counts using the Medium LED setting, it is recommended that the Low setting be used for the assay instead.

  • Page 96: Results Editor

    Results Editor 3.7.1 Post-run analysis main screen At the end of a run, if an analysis method was assigned to a stage with a read, the analysed readings (calculated using either the software defaults or parameters set by the user prior to the run) will be presented in the Results Editor.

  • Page 97: Viewing The Results Of A Run

    • Also found on the results tabs is the Log/Audit Trail tab, which provides details of the run useful for GLP purposes (see section 3.7.5), while the Report tab displays the information sent to the PrimeQ report (see section 3.7.6). C. Combine replicates: Clicking this button combines replicate readings to provide an average for a sample.
  • Page 98 Displays the results table Displays a graph of the standard curve (where applicable). The PrimeQ Report can be printed simply by selecting the Print option within the Report tab or selecting the print icon. Show All Wells: Clicking this button restores the graphical display of data from all wells if...

  • Page 99: Editing A Graph

    the graphs has been maximized as the user does not have to return to the plate layout display to re-select the data. J. Finish/save icons: The results file can be saved at any point during the setup using typical Windows commands. However, if the padlock icon in the top-left of the Results Editor screen is locked, the current file in use is designated read only.

  • Page 100: Changing The Analysis Parameters

    • If more sophisticated editing functions are required, access the Graph Properties… option from the Results Editor menu bar. Select Analysis and then Graph Properties… The graph editing box is equipped with many features that allow the user to edit different aspects of the graph including its appearance, the data itself or the print and export options.
  • Page 101 Clicking this button brings up the analysis parameters relevant to the analysis being performed. The parameters can be edited with changes reflected immediately in the adjacent graph(s). 3.7.4.2 Change the analysis method An entirely different analysis method can be chosen or existing parameters edited from the Analysis Selection box found on the Results Editor main page.

  • Page 102: Log/Audit Trail

    Go to next page/go to last page. 3.7.6 Report The PrimeQ Report can be viewed by clicking on the Report tab in the Results Editor. The results displayed will depend on the report options selected during the analysis method setup (in the Report Options pane of the Analysis Wizard) although default details included in the report are: •...
  • Page 103 Summary of filters used at each stage, emission and excitation wavelengths and user-defined name Integration time • Table of results 3.7.6.1 Report layout 3.7.6.2 Report screen function buttons Frequently used functions are found on the Task bar: Show current page number out of total. Go to first page/previous page.

  • Page 104: Exporting And Printing Results

    3.8.1 Exporting • PrimeQ files: Plate layouts, programs, experiments and results files are all easily transportable as electronic files – simply double-click on the file and it will open straight up into the appropriate program. If transferring multiple files then a file compression program, such as WinZip, will be useful.

  • Page 105: Printing

    Click on Copy to copy the data to the clipboard. The data can then be pasted into Excel. 3.8.2 Printing The PrimeQ Report can be printed simply by selecting the Print option within the Report tab or selecting the Print icon. Individual graphs can be printed by right-clicking with the mouse and selecting Print. Alternatively they can be printed using the Graph Properties option accessed from the Analysis menu bar in the Results Editor (detailed in section 3.7.3).

  • Page 107: Data Analysis

    About this chapter This chapter looks in more detail at the different analysis methods available to users of PrimeQ. Following a brief introduction providing a reminder of the theory behind real-time PCR data collection, the chapter goes on to discuss each analysis in more detail including setup, applications and tips for hands-on use.

  • Page 109: Introduction

    Introduction PrimeQ can be used to determine the absolute or relative quantity of a target DNA template in a given test sample by measuring the cycle-to-cycle change in the fluorescent signal. It can also measure the simple presence or absence of a target. The fluorescent signal is taken to increase...

  • Page 110: Fit Points

    Two approaches are available for calculating the quantification cycle on PrimeQ: fit points and first derivative maximum. 4.1.3 Fit points This approach is the preferred approach when using low copy number templates and is based on the setting of a crossing line and noise threshold.

  • Page 111: First Derivative Maximum

    the early stages is expanded and the Cq value can be determined more accurately. Although some values may be negative when plotted on a log scale (especially if the data has been background corrected) this will not affect the accuracy of the data. 4.1.4 First derivative maximum Simple to perform and requiring no user input, this method determines the quantification cycle by calculating the point on the reaction curve at which the rate of change in fluorescence is fastest.
  • Page 112 and as the Tm is characteristic of the GC content, length and sequence of a DNA product, it is a useful tool in product identification. Dissociation curve showing the dissociation 2,500 peaks. 2,000 1,500 1,000 Tem perature At the start of an analysis, the reaction is at a low temperature such that the DNA in the reaction tube will be double-stranded and the fluorescence signal high.

  • Page 113: Choosing An Analysis Method

    Choosing an analysis method Quansoft allows the user to assign an analysis method to any stage of the PCR that has fluorescent readings. The available analysis methods are: • None: When no analysis method has been set, the Results Editor will display the plate layout and a graph of raw fluorescent data.
  • Page 114 Allelic discrimination (without baseline correction or analysis method options) Two or more Two or more Baseline Quantification (all options) Multi-read Plus/minus (all options) Allelic discrimination (all options) Ramp read Two or more Dissociation curve 4.2.1.1 General points • Analysis methods can be added or changed post-run as long as the assay setup is valid for the method (see above table).

  • Page 115: Analysis Method: None

    Analysis method: None The default analysis method is None, so analysis parameters do not need to be set. 4.3.1 Viewing the results During the run, the real-time collection of data can be monitored in the Run Screen. The plate layout shows the fluorescence curve on a per-well basis and the temperature profile plot indicates how far the run has progressed.

  • Page 116: Primeq Report

    4.3.2 PrimeQ Report The PrimeQ Report shows the plate layout and raw fluorescence data with the default run details displayed by scrolling down the screen. See section 3.7.6.

  • Page 117: Passive Reference Dye (Prd) Correction

    The readings are normalized by dividing the fluorescence of the reporter in each well by the PRD for each well. This correction method is applicable to all the analysis types available on PrimeQ except “None”. Setup procedure:...

  • Page 118: Analysis Method: Baseline Correction

    Analysis method: Baseline correction This method allows the user to adjust the data for any background fluorescence. This approach looks at the signal in the early cycles of the PCR and then averages out the early noise and subtracts it from subsequent readings. As with passive reference dye (PRD) correction detailed above, baseline correction can be useful for correcting the data prior to performing an analysis and so can help to increase the accuracy of the assay.
  • Page 119 4.5.2.2 Baseline correction • Click Next. Options appear for baseline correction. • None: No correction. • Arithmetic 1: Subtracts the average of the specified range of readings from each well. • Arithmetic 2: Subtracts the average of a specified number of lowest readings from each well.

  • Page 120: Viewing The Results

    4.5.2.3 Report Options and Summary • Clicking Next leads through to the Report Options screen. Baseline corrected data can be displayed in a 96-well or a graphical format – click the appropriate box to select. • Click Next to view a summary of the setup. •...
  • Page 121 4.5.3.1 Viewing and changing the parameters • Click the PAR button next to the baseline correction graph to bring up the parameter settings for the analysis. If any settings are changed, the data will be recalculated and the graphs and results table updated accordingly.

  • Page 122: Primeq Report

    4.5.4 PrimeQ Report 4.5.4.1 Display options The report options can be changed from within the report tab of the Results Editor. • Click on the report options icon, which will bring up the Report Options box. Tabs will display the report options relevant for each stage. Change as appropriate and click Done to finish.

  • Page 123: Analysis Method: Quantification

    Analysis method: Quantification This analysis method has two general approaches: either to determine the ‘absolute’ concentration of an unknown sample by comparison of the quantification cycle (Cq) to a standard curve of known concentrations or else a relative value determined by the comparison of Cqs. The latter approach is useful in screening assays, for example, when an increase relative to a control sample provides sufficient experimental information such that an ‘absolute’...
  • Page 124 4.6.2.1 If a PRD was assigned in the dye usage box, the next screen displays an option for passive reference dye correction (see section 4.4). Clicking Next leads through to the Baseline Correction screen. 4.6.2.2 Baseline correction See section 4.5. Choose the method as required and set the values as appropriate. 4.6.2.3 Cq calculation The crossing line is a best-fit of where the reaction efficiency is at its highest and most constant for...
  • Page 125 Setting the noise threshold and crossing line Overview of procedure: The noise threshold and crossing line are placed on the amplification graph either at positions specified by the user or using the default settings. Default values: Noise threshold cursor: n1 = 4 SD above average of all readings x to y Crossing line cursor: n2 = 10 SD above average of all readings x to y x = 3 (or 1 if number of readings less than 10)
  • Page 126 Calculating the quantification cycle Fit points are a defined number of fluorescent points found on the curve just above the crossing line. The points are used in a linear regression calculation and the interception of the linear regression through the fit points and crossing cursor provides a value for the quantification cycle. This will be a fractional number calculated for each well.

  • Page 127: Viewing The Results

    4.6.3 Viewing the results During the run, the real-time collection of data can be monitored in the Run Screen. The plate layout shows the fluorescence curve on a per-well basis and the temperature profile plot indicates how far the run has progressed. When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its own tab.

  • Page 128: Primeq Report

    PAR icon next to the raw data graph. • Click the raw data graph icon if the raw data graph is not displayed. 4.6.4 PrimeQ Report The report options can be changed from within the report tab of the Results Editor. Click on the report options icon, which will bring up the Report Options box.
  • Page 129 Overview of procedure: The experiment is set up using the Quantification Wizard as detailed above. The experiment must include a dilution series of standards in the plate layout by which to compare the unknowns. The concentration of each standard should be added to the Well Information table in the Plate Layout Editor (can be accessed through the Results Editor main screen, post-run).
  • Page 130 4.6.6.2 The standard curve y = -3.379x + 36.724 R² = 0.996 E = 1.977 Log Concentration Linear regression is used to generate a straight line, y = mx + c whereby: • y = the slope, ideally this should be approximately -3.32. •...
  • Page 131 y = -3.402x + 43.278 y = -3.402x + 43.278 y = -3.402x + 43.278 y = -3.402x + 43.278 R² = 0.999 E = 1.967 R² = 0.999 E = 1.967 R² = 0.999 E = 1.967 R² = 0.999 E = 1.967 Log Concentration Log Concentration Log Concentration...

  • Page 132: Comparing Cqs In Relative Quantification

    4.6.7 Comparing Cqs in relative quantification Cqs are also used for relative quantification i.e. the comparison of two different reporters in the same well. Although this approach can be performed either with or without a standard curve, it is particularly useful for screening assays where it is necessary to compare a fold difference of sample B to a calibrator sample A, for example.
  • Page 133 • Select which reporters are to be compared using the drop-down menu. The concentration of the first reporter selected will be divided by that of the second. Clicking Next takes the user through to Report Options window as before. • Click through to the Summary window and click Finish. 4.6.7.2 Viewing relative quantification results During the run, the real-time collection of data can be monitored in the Run Screen.
  • Page 134 The results table displays the calculated results. Column headings: • No: Well number. • Well ID: Location of well. • Sample ID: User-supplied or default name of well. • Cq (Dye 1): Quantification cycle for Dye 1. • Conc (Dye 1): Calculated concentration of sample for Dye 1. •...
  • Page 135 Choosing Relative Cq in the Relative Quantification window brings up the settings box. The user can select which reporters to compare using the drop-down box shown above. The first is usually the gene of interest and the second the REF gene amplified from the same sample. The Cq of the second reporter selected will be subtracted from that of the first and the resulting ∆Cq value can then be compared to that of a calibrator (if a reference sample has been defined as a calibrator, CAL, in the plate layout).
  • Page 136 • Dye to view options offer relative quantification cycles or individual reporter results and graphs. • Changing the Dye to View from Relative Cq to a single reporter displays the baseline corrected graph (if chosen in the setup) in addition to the quantification cycles graph for the individual dyes.

  • Page 137: Quick Guide To Quantification Analysis

    Relative Cq and select which reporter is to be compared to which. Click Next. 5. Report options: select which results should appear in the PrimeQ report. Click Next. 6. Summary of analysis. Click Finish to return to the Experiment or Results Editor main screen.

  • Page 138: Analysis Method: Dissociation Curve

    Analysis method: Dissociation curve Dissociation curve analysis can add to the information obtained from the PCR. Also known as melting curve analysis, it measures the temperature at which the DNA strands dissociate (i.e. the ® melting temperature or Tm). Using an intercalating dye such as SYBR Green I and increasing the temperature in small increments, the PCR products can be seen to ‘unzip’...

  • Page 139: Assay Requirements

    4.7.1 Assay requirements • Need at least one reporter dye. • Need a stage in the program with a ramp read (with at least two readings). 4.7.2 Setup Raw data Raw data Background Background Dissociation Dissociation Dissociation Dissociation Peak areas Peak areas for reporter for reporter...
  • Page 140 Digital filter This is an option to smooth the raw data before it is presented. It is carried out using a Savitsky- Golay curve smoothing algorithm, which fits a third order polynomial to a number of points either side of the data to be smoothed. This is similar to averaging points either side of a centre point but because a third order polynomial curve is used, the profile of the curve is retained.
  • Page 141 A1–A2: These cursors determine the effect of temperature on the fluorescence signal. They should be positioned in the flat fluorescence region measured before any product has melted. The default values are: • A1: Start temperature +10% ramp • A2: Start temperature +30% ramp For example, if the temperature ramps for 30°...
  • Page 142 4.7.2.4 Manual peak detect To use manual peak detect, select this option on the Dissociation Wizard Peak Detector screen. • Number of peaks to find: Up to a maximum of four. • Bin threshold: The user can specify a temperature range (° C) in which a peak will be considered valid.
  • Page 143 Peak Headings Each peak cursor can be individually named by typing a name in the appropriate Peak Headings box. • Click on the PAR button to view the Peak Headings box. • Type in a name for the peak. The new name will then appear in the Results Table column heading and in the Report. This may facilitate identification of peaks corresponding to different PCR products.
  • Page 144 Bin threshold The user can specify a temperature range (ºC) either side of a cursor in which a peak will be considered valid. For example, if the bin threshold is set to 80° C with a range of 0.5° C, then all peaks with a Tm between 79.5 and 80.5°...

  • Page 145: Viewing The Results

    Report Options and Summary • Clicking Next leads through to the Report Options screen, which allows the user to decide how the data appears in the PrimeQ report. • Click Next to view a Summary of the setup. • Click Back to change any settings or Cancel to abort the procedure.

  • Page 146: Primeq Report

    Analysis Selection box from the Results Editor main page. 4.7.4 PrimeQ Report The report options can be changed from within the report tab of the Results Editor. Click on the Report Options icon, which will bring up the Report Options box. Tabs will display the report options relevant for each stage.
  • Page 147 • Peak area: Check the box to have peak area calculated from the data. Click Next. • Report options: Decide which data to display in the PrimeQ report. Click Next. • Summary of analysis. Click Finish to return to the Experiment or Results Editor main page.

  • Page 148: Analysis Method: Plus-Minus Scoring

    Analysis method: Plus-minus scoring This type of assay is used to record the presence or absence of a PCR product. Input data can either be kinetic (whereby the number of readings is >1) or end-point (a reading taken at the end of the run).
  • Page 149 4.8.2.1 Baseline correction This option is only available if the data is kinetic (i.e. > 1 reading). The choice of baseline correction method is similar to that shown in the Baseline Correction Wizard (section 4.5) with the exception that Proportional is excluded. Click Next when the baseline correction is set. 4.8.2.2 Plus-minus analysis method This screen allows the user to choose which readings are to be used for results scoring.

  • Page 150: Viewing The Results

    4.8.2.4 Report Options and Summary • Click Next to lead through to the Report Options screen. The default is for the plus/minus table to be displayed but an option for a scaled data graph displaying the confidence thresholds is also available. •...
  • Page 151 Clicking an individual well or a selection of wells in the plate layout will highlight just the selected well(s) on the plus-minus graph. Clicking the Show all wells button will re-select all wells. The plus-minus graph Well If the plus-minus graph is not displayed, click on the icon to bring up the data: The plus-minus graph displays the values as points and the confidence threshold as sliding cursors (blue is the lower threshold and red, the upper).

  • Page 152: Primeq Report

    If the raw data graph is not shown click the raw data graph icon: 4.8.4 PrimeQ Report The report options can be changed from within the report tab of the Results Editor. Click on the Report Options icon, which will bring up the Report Options box. Tabs will display the report options relevant for each stage.
  • Page 153 5. Choose a plus-minus analysis method and specify a range of readings if necessary. Click Next. 6. Set upper and lower confidence thresholds. Click Next. 7. Report options: Decide how to display the data in the PrimeQ report. Plus-minus score table is the default setting but choose to display in graphical format. Click Next.

  • Page 154: Analysis Method: Allelic Discrimination

    Allelic discrimination on the PrimeQ real-time PCR system allows these stages to be performed automatically within the reaction tube, thereby providing an assay which is rapid, reliable and cost-effective in terms of both reagents and time.
  • Page 155 • Click Next and the Allelic Discrimination Wizard will launch. 4.9.2.1 Baseline correction The baseline correction methods are the same as for plus-minus scoring in that the data should be kinetic (i.e. > 1 reading) and that the Proportional baseline correction method is unavailable. •...

  • Page 156: Viewing The Results

    4.9.2.3 Reporter selection The fluorescence of the first reporter is plotted against the second and displayed on a scatter graph. 4.9.2.4 Report Options and Summary • Click Next to lead through to the Report Options screen. The default is for an allelic discrimination graph and a table of results.
  • Page 157 4.9.3.1 Allelic discrimination scatter graph Particular to the allelic discrimination method is the display of a scatter plot representing the fluorescence of one allele plotted against the other. The software plots the results of the run on a scatter plot of allele 1 vs. allele 2 (axes defined by the user in Reporter Selection) with each well of the 96-well plate represented by a point on the graph.
  • Page 158 copies of both alleles and therefore produce fluorescence with both dyes. Samples that do not cluster tightly may contain rare sequence variations, sequence duplications or the PCR may not have been optimal. Assigning Genotypes The user can define clusters of points within the plot that represent genotypic segregation within the samples;...

  • Page 159: Primeq Report

    Analysis Selection box from the Results Editor main page. 4.9.4 PrimeQ Report The report options can be changed from within the report tab of the Results Editor. Click on the Report Options icon, which will bring up the Report Options box. Tabs will display the report options relevant for each stage.
  • Page 160 Click Next. 6. Report options: Decide which data should be displayed in the PrimeQ report. A table of results is the default setting but choose to display the data in a graphical format if required. Click Next.

  • Page 161: Analysis Method: Multi-Read

    Cq values obtained from quantification analysis of the amplification stage and used to aid primer optimization experiments. Alternatively, Multi-Read allows PrimeQ to be used simply as a plate reader.

  • Page 162: Viewing The Results

    • End-point: Uses the last reading only (we recommend that >1 reading is used for accuracy). • All readings (default): Averages all readings in the stage • Last readings: Averages a user-specified number of last readings (the default is 5, or 1 if there are less than 5 readings) •...
  • Page 163 When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its own tab. Multi-read graph The graph displays the average readings for each well as colour-coded points. If the multi-read graph is not displayed, simply click on the Multi-read icon to bring up the data: •...

  • Page 164: Primeq Report

    If the raw data graph is not shown click the raw data graph icon: 4.10.4 PrimeQ Report The report options can be changed from within the report tab of the Results Editor. Click on the Report Options icon, which will bring up the Report Options box. Tabs will display the report...

  • Page 165: Quick Guide To Multi-Read Analysis

    4.10.5 Quick guide to multi-read analysis 1. In the Experiment or Results Editor Analysis Selection box, highlight the stage on which multi- read analysis is to be performed and click Edit. 2. In the Analysis Wizard Selection box, choose Multi-Read from the drop-down menu and assign a use next to the appropriate dye(s) name.

  • Page 166: Multiplex Assays

    Multiplex assays (or multi-colour detection) combine the use of two or more different reporters in the same well. PrimeQ’s cartridge carousel can house up to four pairs of excitation and emission filters, and as such, is capable of detecting up to four different reporters.
  • Page 167 4.11.1.1 Viewing the results • Dye to View: Use this option to view the data for the individual reporters. • Results table: Results for only the dye currently being viewed will be listed in the results table unless an analysis method has been selected where more than one reporter is included in the calculation e.g.

  • Page 169: Technical Information

    5 Technical information Technical information on PrimeQ About this chapter This chapter provides all the technical information you may need to know about your PrimeQ real-time PCR system. It covers everything from spare parts to recommended consumables and maintenance routines.

  • Page 171: Operator Maintenance

    Please include any details of the fault observed and remember to return the unit in its original packing. Techne accept no responsibility for damage to units which are not properly packed for shipping: if in doubt, contact your supplier giving the full serial number of the unit and the firmware version number (shown on the LCD screen when the unit is first switched on).

  • Page 172: Insulation Testing

    Indique de forma detallada todos los defectos que haya notado y devuelva el equipo en su embalaje original. Techne no aceptará responsabilidad alguna por daños causados en equipos que no estuvieran debidamente embalados para su envío; si tuviera alguna duda, póngase en contacto con su proveedor.
  • Page 173 La protection de l’appareil est assurée par deux fusibles dont le remplacement ne peut être effectué que par un personnel qualifié. Si les fusibles sautent sans arrêt, il s’agit d’un problème sérieux. Nous vous conseillons dans ce cas de prendre contact avec votre fournisseur pour réparation...

  • Page 174: Block Access

    Then slide the quick release handles in the opposite direction (closed padlock position) to lock the block assembly in position. The thermal blocks are internally calibrated so when replaced, temperature performance is unchanged and recalibration is not required. The PrimeQ unit is guaranteed for a period of 2 years from the date of purchase.

  • Page 175: User Responsibilities

    96-well clear plates. Black plates give the lowest background, and white plates, although they have the highest background, may be advantageous for some probe-based chemistries. PrimeQ does not require a specific seal as long as the seal of choice is compatible with the unit’s optical reads. further...

  • Page 176: Minimum Computer Requirements

    Ethernet connection ® Software: Internet Explorer, Microsoft Office Accessories The following accessories can be obtained from Techne or your Techne dealer. Please note that additional filters may be available upon request: Part number Description Unit quantity FC01 Filter cartridge for FAM multiplex ®...

  • Page 177: Replacement Parts

    Replacement parts The following replacement parts can be obtained from Techne or your Techne dealer: Part number Description Quantity required HH179(S) UK mains lead with plug HH180(S) EU mains lead with plug 7002705 US Power cord and 3-pin plug 120V...

  • Page 178: Packing The Primeq Instrument

    • Wrap each filter in the tissue paper and place in the bag with the silica gel sachet. Replace in the filter cartridge storage box. There is a space in the PrimeQ carton for the cartridge box, between the foam instrument surrounds.

  • Page 179: Packing The Instrument

    This prevents the drawer from moving against its latch during transit, which could cause wear. • Pack the block and filters; wedge the drawer: • Bag the PrimeQ and place into the carton base: • Add foam retainers and the block and filter boxes:...

  • Page 180: Packaging

    Techne takes no responsibility for returned goods damaged in transit. If you do not have the original packing you can purchase a new pack from your dealer, part number 5100492.

  • Page 181: Troubleshooting

    6 Troubleshooting Troubleshooting real-time PCR and PrimeQ About this chapter This chapter contains information required for troubleshooting the real-time PCR process, the control software and the instrument itself.

  • Page 183: Troubleshooting

    Troubleshooting ISSUE CAUSE SOLUTION No display on LCD No power to instrument. Check power supply is connected. screen. Fuse blown in plug. Check fuse and change if appropriate. No amplification. Incorrect reaction conditions. Optimize assay and run agarose gel to check PCR Reaction component not Check correct reagents were added.
  • Page 184 ISSUE CAUSE SOLUTION No Cq recorded for No template added. Repeat assay. a sample. No target in sample. Noise and crossing line set The default settings in Quansoft are 4 incorrectly. and 10 standard deviations respectively, adjust these through the parameter option or the Analysis Wizard.
  • Page 185 Cartridge Access and Editing screen. Fatal error displayed Instrument error. Note the error message and contact your on LCD screen. Techne Distributor. Any other error Instrument error. Open Windows Explorer. which prevents a Open C:\Program PCR run being Files\Techne\PrimeQIM\Data initiated.

  • Page 186: Real-Time Pcr Glossary

    Complementary DNA produced by reverse transcription of total or messenger RNA. Quantitation cycle, the cycle number at which the fluorescence increases past a fixed threshold. PrimeQ has default values of 10 standard deviations above the noise for the threshold or Crossing Line.
  • Page 187 PCR Efficiency An efficient PCR assay shows a doubling in the specific product after every cycle: -1/slope Reaction efficiency E = 10 ideal value is 2 The efficiency is affected by the length, GC content and secondary structure of the amplicon and sub-optimal reaction conditions. Passive Reference Dye: Internal reference dye to which the reporter dye is normalized to correct for changes in volume and block variations.

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