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BIO RAD CFX384, Instruction Manual

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BIO RAD CFX384 Instruction Manual Download Page 39

Running Experiments

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Summary of Contents for CFX384

Page 1: ...CFX96 and CFX384 Real Time PCR Detection Systems Instruction Manual Catalog 184 5384 185 5384 184 5096 185 5096 ...

Page 2: ...licensed under U S Patent No 6 814 934 and corresponding claims in any Canadian counterpart patent thereof owned by Applera Corporation for use solely in research and all applied fields except human and veterinary in vitro diagnostics These license rights are effective only if this detection module is combined with a Bio Rad thermal cycler for which the applicable real time thermal cycler royalty ...

Page 3: ...d website by selecting your country on the home page www bio rad com Find the nearest international office listed on the back of this manual Technical notes and literature Go to the Bio Rad website www bio rad com or Gene Expression Gateway www bio rad com genomics Type a search term in the Search box and select Literature to find links to technical notes manuals and other literature Technical spe...

Page 4: ...r information before proceeding CAUTION Hot surface This symbol identifies components that pose a risk of personal injury due to excessive heat if improperly handled Table 4 Instrument Safety Warning Labels Icon Meaning Warning about risk of harm to body or equipment Operating the CFX96 or CFX384 real time PCR detection system before reading this manual can constitute a personal injury hazard For ...

Page 5: ... A1 EN61010 2 081 2002 A1 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 081 Particular requirements for automatic and semi automatic laboratory equipment for analysis and other purposes includes Amendment 1 EN 61326 1 2006 Class A Electrical equipment for measurement control and laboratory use EMC requirements Part 1 General requirements This equipm...

Page 6: ...er Software 6 Running Experiments 9 Chapter 2 Introduction to CFX Manager Software 11 Main Software Window 11 Startup Wizard 15 Detected Instruments Pane 16 Instrument Properties Window 18 Software Files 20 Tips and Tricks 20 Chapter 3 Running Experiments 21 Experiment Setup Window 21 Protocol Tab 22 Plate Tab 23 Start Run Tab 24 Run Details Window 25 Instrument Summary Window 28 Chapter 4 Protoco...

Page 7: ...Analysis Menu Bar 71 Quantitation Tab 72 Data Analysis Settings 73 Well Selectors 75 Charts 78 Spreadsheets 79 Chapter 8 Data Analysis Windows 81 Quantitation Tab 81 Quantitation Data Tab 85 Melt Curve Tab 87 Melt Curve Data Tab 89 End Point Tab 91 Allelic Discrimination Tab 93 QC Tab 95 Reports for Data Files 97 Chapter 9 Gene Expression Analysis 101 Gene Expression 101 Plate Setup for Gene Expre...

Page 8: ...Tab 123 Files Tab 124 Protocol Tab 124 Plate Tab 125 Data Analysis Tab 126 Gene Expression Tab 127 QC Tab 128 User Administration 129 Chapter 11 Resources 131 Calibration Wizard 131 Instrument Maintenance 133 Application Log 135 Software Help Tools 135 Troubleshooting 136 Instruments Parts and Accessories 138 References 139 Index 141 ...

Page 9: ...Table of Contents viii ...

Page 10: ...ager software uick guides for system installation protocol plate data analysis and gene expression analysis Remove all packing materials and store them for future use If any items are missing or damaged contact your local Bio Rad office System Requirements To operate the CFX96 or CFX384 system use the following power sources and cables Input power 100 240 VAC 50 60 Hz Indoor use Ambient temperatur...

Page 11: ...rmal cycler base The C1000 base includes a user interface to control the system when running in stand alone mode and the power button and ports both on back panel to connect to a computer Figure 1 Front view of the CFX96 system When open the CFX96 or CFX384 system includes the features shown in Figure 2 Figure 2 Inside view of the CFX96 system WARNING Avoid touching the inner lid or block These su...

Page 12: ... here Ethernet port Connect an ethernet cable to email run logs and stand alone data files USB connections Use these ports to connect the CFX96 system or CFX384 system to a computer or to connect an S1000Tm thermal cycler Figure 3 Back panel of C1000 thermal cycler WARNING Avoid contact with the back panel of the C1000 during operation Setting up the system The CFX96 or CFX384 real time PCR detect...

Page 13: ...tion module bay of the C1000 chassis leaving about 2 cm of space in the front When in the chassis bay the optical module should be covering the Bio Rad logo in front of the bay of the C1000 chassis 4 Reach around and pull up the locking bar of the C1000 until it is flush with the sides of the module bay This action moves the module forward locking it into place Figure 5 Figure 5 Locking the optica...

Page 14: ...and C1000 base is uneven reinstall the module starting from Step 2 page 4 6 Plug the power cord into the back of the C1000 base Figure 3 on page 3 and into an appropriate three pronged electrical outlet 7 Press the power switch on the back panel of the C1000 thermal cycler to start the system 8 Follow the instructions in the C1000 front panel to remove the red shipping screw from the inner heater ...

Page 15: ...oftware on a PC computer with a Windows 64 bit Operating Systems is not supported due to incompatible USB Drivers A PC computer with a 64 bit processor like Intel on a 32 bit Windows Operating System is supported To install CFX Manager software 1 The software must be installed on the computer by a user with administrative privileges Make sure you are logged in with administrative privileges 2 Plac...

Page 16: ...n the Readme txt file See Installing the Software Manually on page 136 Installing the Drivers If the CFX96 system or CFX384 system is going to be run in Software controlled mode drivers must be installed on the computer Use only the supplied USB cable which is sufficiently shielded to prevent data loss To install the system drivers 1 Connect the C1000 chassis to the computer by plugging a USB cabl...

Page 17: ...date to search for software Figure 9 Click Next Figure 9 Found New Hardware Wizard 4 Instruct the wizard to Install the software automatically Click Next to continue installing the drivers Figure 10 Figure 10 Software Driver installation screen 5 Click Finish at the software installation completion screen when the drivers are installed ...

Page 18: ... 8 tube strips without caps white wells TCS 0803 Optical flat 8 cap strips for 0 2 ml tubes and plates MSB 1001 Microseal B adhesive seals optically clear Loading the Block To load your reactions in the block follow these suggestions Click the Open Lid button located on software s Start Run tab see Start Run Tab on page 24 or press the lid button on the front of the system Figure 1 to start openin...

Page 19: ...empty tube strip with caps on the right side of the block to balance the pressure applied by the heated lid Figure 12 Balance the tube strips or cut microplates in the block WARNING Be sure that nothing is blocking the lid when it closes Although there is a safety mechanism to prevent the lid from closing if it senses an obstruction do not place anything in the way of the closing lid Well H3 Well ...

Page 20: ...s that ships with the system Installation quick guide Protocol quick guide Plate quick guide Data Analysis quick guide Gene Expression Analysis quick guide TIP See the software Help for more guides about running experiments Get started in the main software window by using these features Figure 13 Status bar View the current software and instrument status page 12 Menu bar Select software commands p...

Page 21: ... Figure 14 to see the current status of instruments Figure 14 Left side of status bar in main software window View the right side of the status bar Figure 15 to see the current user name date and time Figure 15 Right side of status bar in the main software window Click and drag the right corner of the status bar to resize the main window Menu Bar The menu bar of the main software window provides t...

Page 22: ...ide the Detected Instruments pane Toolbar Show or hide the main software window toolbar Status Bar Show or hide the main software window status bar User Select User Open the Select User window to change software users Change Password Change your user password User Preferences Open the User Preferences window User Administration Manage users in the User Administration window Tools Dye Calibration W...

Page 23: ...Website View a website that lists Bio Rad consumables for PCR and real time PCR reagents PCR Plastic Consumables Website View a website that lists Bio Rad consumables for PCR and real time PCR experiments Software Updates Check for software updates from Bio Rad About Open a window to see the software version Table 8 Toolbar buttons in the main software window Button Button Name Function Open a Dat...

Page 24: ...g Experiment Setup Open the Experiment Setup window to run an experiment page 21 Protocol AutoWriter Open the Protocol AutoWriter window to create a new protocol page 37 Select User Open the Select User window to change software users see Log in or Select User on page 121 User Preferences Open the User Preferences window page 121 Help Open the software Help window for more information about runnin...

Page 25: ...seconds 96 well fast reaction module gradient enabled 384 well reaction module Dual 48 48 fast reaction module gradient enabled S1000 thermal cycler connected to a C1000 thermal cycler For more information see S1000 and C1000 thermal cycler instruction manuals TIP Locate PDF copies of the instruction manuals by opening the Documents folder on the CFX Manager software installation CD You can simult...

Page 26: ...nt Properties Open the Instrument Properties window Collapse All Collapse the list of instruments in the Detected Instruments pane Expand All Expand the list of instruments in the Detected Instruments pane You can also control a block by clicking an instrument block icon in the Detected Instrument pane and then clicking a button in the Selected Instrument pane Figure 19 Figure 19 Buttons at the bo...

Page 27: ...want to transport the instrument Calibrated Dyes View the list of calibrated fluorophores Figure 20 Instrument Properties window Properties Tab The default name for an instrument is the C1000 thermal cycler serial number which appears in many locations including the Detected Instruments pane Figure 18 To rename an instrument for ease of identification follow these instructions In the Instrument Pr...

Page 28: ...tab in front Follow the instructions to remove the screw The information in this tab changes depending on whether the shipping screw is installed or removed For example to install the shipping screw click the Install Shipping Screw button and follow the instructions in the tab Figure 21 Figure 21 Instructions for installing the shipping screw Calibrated Dyes Tab Open the Calibrated Dyes tab to vie...

Page 29: ...te files from the Express Load menu add or delete the files prcl and pltd extensions in the ExpressLoad folder select Tools User Data Folder in the menu bar of the main software window To view all the information loaded into one well in a plate double click the well to open the Well Info window Right click any graph or chart to change viewing and data analysis options Edit well contents before dur...

Page 30: ...ain software menu bar page 12 The Experiment Setup window includes three tabs Protocol Click the Protocol tab to select an existing protocol to run or edit or to create a new protocol in the Protocol Editor window page 31 Plate Click the Plate tab to select an existing plate to run or edit or to create a new plate in the Plate Editor window page 41 Start Run Click the Start Run tab page 24 to chec...

Page 31: ... Create New button Open the Protocol Editor to create a new protocol Select Existing button Open a browser window to select and load an existing protocol file prcl extension into the Protocol tab Express Load pull down menu Quickly select a protocol to load it into the Protocol tab TIP To add or delete protocols in the Express Load menu add or delete files prcl extension in the ExpressLoad folder ...

Page 32: ...scription of the contents of each well the scan mode and the plate type CFX Manager software uses these descriptions for data collection and analysis Select one of the following options to select an existing plate create a new plate or edit the currently selected plate Create New button Open the Plate Editor to create a new plate Select Existing button Open a browser window to select and load an e...

Page 33: ...tarted including the selected protocol and plate files and a section for selecting the instrument block Run Information pane View the selected Protocol file Plate file and data acquisition Scan Mode setting Enter optional notes about the experiment in the Notes box Start Run on Selected Block s pane Select one or more blocks edit run parameters if necessary and then click the Start Run button to b...

Page 34: ... Start Run tab to remotely operate the selected instruments Start Run Start the experiment on the selected instrument blocks Flash Block Indicator Flash the indicator LED on the selected instrument blocks Open Lid Open motorized lid on selected instrument blocks Close Lid Close motorized lid on selected instrument blocks Run Details Window When you click the Start Run button CFX Manager software p...

Page 35: ...buttons Click one of the buttons to remotely operate the instrument or to interrupt the current protocol Run Information pane Displays experiment details Run Status Tab Buttons Click one of the buttons listed in Table 11 to operate the instrument from the software or to change the run that is in progress NOTE Changing the protocol during the run such as adding repeats does not change the protocol ...

Page 36: ...igure 29 The Real time Status tab displays the data during a run Add more repeats to the current GOTO step in the protocol This button is only available when a GOTO step is running Skip the current step in the protocol If you skip a GOTO step the software verifies that you want to skip the entire GOTO loop and proceed to the next step in the protocol Flash the LED on the selected instrument to ide...

Page 37: ...ress Load folder Time Status Tab The Time Status tab shows a countdown timer for the current run Figure 30 Figure 30 The Time Status tab displays a count down timer for the current run Instrument Summary Window The Instrument Summary window shows a list of the detected instruments and their status Open the Instrument Summary by clicking the View Summary button Figure 19 on page 17 in the Detected ...

Page 38: ...ame Function Set Up Experiment Set up an experiment on the selected block by opening the Experiment Setup window Stop Stop the current run on selected blocks Pause Pause the current run on selected blocks Resume Resume the run on selected blocks Flash Block Indicator Flash the indicator LED on the lid of the selected blocks Open Lid Open a selected block motorized lid Close Lid Close a selected bl...

Page 39: ...le Print Print the list Print Selection Print only the selected cells in the list Export to Excel Export the list as an Excel formatted file Export to Text Export the list as a text file Find Find text in the list Sort Sort the list by selecting up to three columns of data in the Sort window ...

Page 40: ... open the Protocol Editor follow one of these options To create a new protocol select File New Protocol or click the Create New button in the Protocol tab page 22 To open an existing protocol select File Open Protocol or click the Open Existing button in the Protocol tab page 22 To edit the current protocol in the Protocol tab click the Edit Selected button in the Protocol tab page 22 TIP To chang...

Page 41: ...mportant functions Figure 33 Protocol Editor toolbar Table 13 Protocol Editor menu bar Menu Item Command Function File Save Save the current protocol Save As Save the current protocol with a new name or in a new location Close Close the Protocol Editor Settings Lid Settings Open the Lid Settings window to change or set the Lid Temperature Tools Gradient Calculator Select the block type for a gradi...

Page 42: ...e step before or after the currently selected step 1 Click the Insert Step button 2 Edit the temperature or hold time by clicking the default value in the graphic or text view and entering a new value Table 14 Protocol Editor toolbar buttons Toolbar Button and Menus Name Function Save Save the current protocol file Print Print the selected window Insert Step Select After or Before to insert steps ...

Page 43: ...tep In Figure 34 notice that the camera icon in the graphic view top shows that step 4 includes a plate read Insert Gradient Button To insert a gradient step before or after the currently selected step 1 Insert a temperature gradient step by clicking the Insert Gradient button 2 Make sure the plate size for the gradient matches the block type of the instrument 96 well or 384 well Select the plate ...

Page 44: ... Figure 35 shows an inserted GOTO step at the end of the protocol Notice that the GOTO loop includes steps 2 through 4 Insert Melt Curve Button To insert a melt curve step before or after the selected step 1 Click the Insert Melt Curve button 2 Edit the melt temperature range or increment time by clicking the default number in the graphic or text view and entering a new value Alternatively click t...

Page 45: ...old enter zero 0 00 for the time Figure 37 shows the selected step with a gradient of 10o C Notice that some options are not available in a gradient step A gradient step cannot include an increment or ramp rate change Figure 37 Step option for a gradient NOTE A gradient runs with the lowest temperature in the front of the block row H and the highest temperature in the back of the block row A The S...

Page 46: ...one of two temperature control modes to determine when the sample reaches the target temperature in a protocol TIP The sample volume can be changed before a run by editing the Sample Volume parameter in the Start Run tab see Start Run Tab on page 24 Enter a sample volume in the protocol editor to select a temperature control mode Calculated mode When you enter a sample volume between 1 and 50 μl 9...

Page 47: ...PCR product Enzyme Select the DNA polymerase enzyme iTaq iProof or Other iTagTm DNA Polymerase iProofTm high fidelity DNA Polymerase enter additional information including hot start activation time and final extension time Run time and type Enter a speed Standard Fast Ultrafast to adjust the total run time and select the type of PCR Real time PCR or PCR The run time for any protocol is influenced ...

Page 48: ... button on the toolbar to open the Protocol AutoWriter window 2 Enter the Annealing Temperature Ta and Amplicon Length in the boxes within the Enter Target Values Enzymes pane If you do not know the annealing temperature for primers click the Ta Calculator button to enter the primer sequences and calculate the annealing temperature For information about the calculations used in the Ta Calculator s...

Page 49: ...Protocols 40 ...

Page 50: ...xperiment Setup window and run it To run a real time PCR experiment you must load the minimal required information in the Plate Editor at least one well must contain a loaded sample type and fluorophore TIP Change the well contents before during and after running the experiment However the scan mode and plate size cannot be changed during or after the run Open the Plate Editor To open the Plate Ed...

Page 51: ...Loading Guide Open the Plate Loading Guide window from the toolbar for a quick overview of instructions to load a plate page 42 Plate view View the current well contents Load wells by using the plate loading options on the right side of the plate view Figure 39 Well loading controls Choose the contents to load in the wells page 46 from the controls on the right side of the plate view The Plate Edi...

Page 52: ...n the plate that holds your samples including BR White and BR Clear For accurate data analysis the plate type must be the same as the plate well type used in the experiment NOTE You must calibrate new plate types page 131 Number Convention Select or cancel the selection for Scientific Notation Units Select the units to show in the spreadsheets when performing quantitation of unknowns versus a stan...

Page 53: ...calibrated for the selected plate type NOTE CFX96 and CFX384 instruments are factory calibrated for many fluorescent dye and plate combinations Calibration is specific to the instrument dye and plate type To calibrate a new combination of dye and plate type on an instrument select Tools Calibration Wizard see Calibration Wizard on page 131 Table 16 Toolbar items in the Plate Editor Toolbar Item Na...

Page 54: ...r NOTE The fluorophores listed depend on the scan mode when SYBR FAM only is chosen only channel 1 fluorophores are shown in the Select Fluorophores window NOTE You cannot add or remove fluorophores in this list you must calibrate the new fluorophores on an instrument in the Calibration Wizard page 131 After calibration the new fluorophore is added to the Select Fluorophore window Click the Select...

Page 55: ...e Names Libraries in the Plate tab in the User Preferences window page 125 Select a well to load contents into by left clicking with the mouse pointer in the plate view Hold down the mouse button and drag to select multiple wells The buttons and lists on the right side of the plate view include all the options needed to load the wells Table 17 Table 17 Options for loading the plate and wells in th...

Page 56: ...ick the Replicate Series button In the Replicate Series pane you can apply a replicate series to a set of selected wells Enter the Replicate Group Size by selecting a number that represents the number of samples wells in each group of replicates Select a Starting Replicate to add replicates NOTE You can load replicate groups with replicate numbers progressing from left to right Horizontal or progr...

Page 57: ...creases Finally select the fluorophore used for the dilution series from the pull down menu and click Apply Select Tools Show Well Notes to show this pane Enter notes about one or more wells by selecting the wells and typing the notes in the pull down menu Any notes you add appear in the spreadsheet on the Quantitation Data tab page 78 Select Tools Show Collection Name to show this pane Enter samp...

Page 58: ...e Targets tab with the analysis settings shown Figure 41 Targets tab in Experiment Settings window Figure 42 shows the Sample Tab with the Analysis Settings shown Figure 42 Samples tab in Experiment Settings window To adjust the lists in these tabs use the following functions Add a target or sample name by typing a name in the New box and clicking Add Remove a target or sample name from the list b...

Page 59: ...to be analyzed in one of four configurations defined by the Sample Name Grouping Option These options are available from the pull down menu in the Experiment Settings tab Target vs Sample Target vs Collection Target vs Sample_Collection Target vs Collection_Sample Well Groups Manager Window Well groups divide a single plate into subsets of wells that can be analyzed independently in the Data Analy...

Page 60: ...to finish and close the window or click Cancel to close the window without making changes Figure 43 Color of wells in the Well Group Manager window Plate Spreadsheet View Window The Plate Spreadsheet View window shows the contents of a plate in the Plate Editor Open the Plate Spreadsheet View window Figure 44 by selecting Tools Show Spreadsheet View in the Plate Editor menu bar Figure 44 Plate Spr...

Page 61: ...ening the Plate Editor and selecting Settings Units in the menu bar After the plate runs the data from these standards appear in the Standard Curve chart of the Quantitation tab Data Analysis window with the units you select Open the spreadsheet view to import or export the plate contents to Excel or another tab delimited format Right click on the spreadsheet to select one of these options from th...

Page 62: ...nel The control panel on the C1000 thermal cycler provides access to all the functions needed to run the instrument utilizing the following three components Liquid crystal display LCD Displays the main menu and other screens Keypad Navigate screens and enter commands with these keys USB port Connect a USB drive mouse or keyboard Figure 45 shows the components of the control panel Figure 45 Thermal...

Page 63: ...ions of keys on control panel Key Function COMMAND KEYS RUN Select and run a protocol EDIT Select and change protocol STATUS View the status of one or more running protocols VIEW Switch between graphic and text view of a protocol FUNCTION KEYS F1 F2 F3 or F4 Function key buttons names and functions change on each screen ALPHANUMERIC KEYS 1 through 9 Enter numbers or letters of the alphabet Press a...

Page 64: ...menu New Protocol F4 Create a new protocol Experiment Setup The CFX96 system or the CFX384 system can run real time PCR experiments without a computer You can export the fluorescence data acquired during a run using the USB thumb key You can also choose to have the data emailed directly to you if the C1000 base is attached to the internet and the email functionality has been configured see Exporti...

Page 65: ...ature step press the arrow keys to navigate between steps and to select a parameter temperature or time Press the alphanumeric keys to enter a new number for each parameter you highlight TIP Connect a computer mouse via a USB port on the C1000 chassis to navigate NOTE Press the VIEW key to switch between graphic and text view of the protocol 3 Optional To insert a new step select the Insert F1 but...

Page 66: ... default sample volume select the sample volume box Vol Figure 47 on page 56 Use the alphanumeric keys to enter a new sample volume in microliters The sample volume you enter determines the temperature control mode that is used during a run TIP Entering a sample volume from 1 to 50 selects Temperature Control mode which is the standard mode Entering zero 0 selects Block mode Temperature mode is th...

Page 67: ...one so or edit the name previously created in the Protocol window Use arrow keys to select a destination folder Figure 49 Figure 49 Saving a protocol 3 Click Edit Filename F1 and type a new name in the box Figure 50 Figure 50 Entering a protocol name 4 Click Save F2 Figure 50 ...

Page 68: ... Figure 51 Figure 51 Protocol successfully saved 6 Edit the Sample Volume or Lid Temperature that will be used for the run Figure 52 7 Enter the Sample ID or User to be recorded in the Run information screen Figure 52 Editing sample volume and lid temperature 8 Click OK F1 to proceed ...

Page 69: ...rrow keys to navigate to the Data File Name box then press the alphanumeric keys to enter a letter or number to type a new data file zpcr name 11 Click the OK F1 button to start the run Running a Previously Saved Protocol To change an existing protocol press the EDIT key to open the file library and select a protocol to edit To run an existing protocol press the RUN command key and select a previo...

Page 70: ...he SYSTEM folder Figure 55 Figure 55 RT_DATA folder stores real time PCR runs Data can be transferred to the analysis computer using a USB key or it can be emailed directly to the computer using the SMTP server once an email connection is configured NOTE The C1000 stores up to 20 real time PCR experiment runs Exporting Data Using the USB Key If a USB key has been placed in a USB key port on the C1...

Page 71: ...a zpcr to the USB key as shown in Figure 56 Figure 56 Exporting stand alone run data to a USB key 5 Use the arrow keys to navigate to the folder on the USB in which to save the file 6 Click Yes F1 to confirm the export Figure 57 Figure 57 Confirming export to USB key Exporting Data Using Email You can choose to email your data to you directly from the C1000 thermal cycler after the run completes b...

Page 72: ...ng the Run command key select Options F4 in the Run information screen Figure 58 Figure 58 Run information screen 2 Using the arrow keys to select the Send email notification option Figure 59 Figure 59 Selecting email notification 3 Click OK F1 to return to the Run information screen 4 Use the arrow keys to navigate to the Email Address box and then use the alphanumeric keys to enter the name of a...

Page 73: ...d Follow these instructions to create a data file from a stand alone run 1 Click and drag the zpcr file from the USB key directory over the main software window or Select File Open Stand alone Run from the main software window menu options to select the file name 2 In the Run File Processor window click the Select Plate button to import the name of the plate file the software will use to create th...

Page 74: ...ager software To configure the outgoing email from the C1000 thermal cycler follows these instructions 1 Connect an ethernet cable to the port in the back of the C1000 chassis 2 On the main menu select Log In F1 to log in to the thermal cycler as the administrator Figure 46 on page 55 NOTE The logged in user name appears under the date and time when you return to the main window 3 Select Utilities...

Page 75: ...twork administrator for your the SMTP server name NOTE The SMTP server name is provided by your ISP 7 Select Add Server Name F1 Figure 64 Figure 64 SMTP server setup window 8 Type the name of the server in the text box using the virtual keypad NOTE The SMTP server name will use the following nomenclature SMTP YourInstitution com Do not use Bio Rad com in the name ...

Page 76: ... the SMTP server Figure 65 Figure 65 Saving server name 10 The added server name will appear in the SMTP Server Names pull down menu as shown in Figure 66 Figure 66 Added server name 11 Select Set Current Server F3 to set the current server to be used for email Figure 66 ...

Page 77: ...t server 14 The C1000 thermal cycler will send an email to the entered address as a test of the SMTP server configuration NOTE Some SMTP servers do not allow attachments and others allow attachments only up to certain sizes If you will use CFX Manager software or the C1000 chassis to email data files and or reports you may want to test your server s ability to email attachments by checking the Tes...

Page 78: ... the Edit View Plate button at the top of the Data Analysis window TIP You can add or edit information about the contents of the well before during or after you run the real time PCR experiment You must assign the scan mode and plate size before the run and these parameters cannot change after the run CFX Manager software processes real time PCR data automatically at the end of each run and opens ...

Page 79: ...ysis Toolbar The toolbar in the Data Analysis window Figure 70 provides quick access to important data analysis functions Figure 70 Toolbar in the Data Analysis window Table 19 lists the functions of buttons in the toolbar Table 19 Toolbar in the Data Analysis window Toolbar button Name Function Save Save the current data file Print Print the selected window Trace Style Open Trace Style window Rep...

Page 80: ...ode to determine how C t values are calculated for each trace Baseline Thresholds Open the Baseline Thresholds window to adjust the baseline or the threshold Trace Styles Open the Trace Styles window View Edit Plate Open the Plate Editor to view and edit the plate Mouse Highlighting Turn on or off the simultaneous highlighting of data with the mouse pointer TIP If the Mouse Highlighting is turned ...

Page 81: ... Data Analysis window NOTE The software links the data in the panes of each data analysis tab For example highlighting a well by placing the mouse pointer over the well in the well selector view highlights the data in all the other panes Step Number Selector The CFX96 system or CFX384 system can acquire fluorescence data at multiple protocol steps the software maintains the data acquired at each s...

Page 82: ... wells in that well group appear loaded with content in the well selector and data only for these wells are included in the data analysis calculations Figure 74 Data Analysis window with Group 2 selected Data Analysis Settings The Amplification chart data in the Quantitation tab shows the relative fluorescence RFU for each well at every cycle Each trace in the chart represents data from a single f...

Page 83: ...e Settings The software automatically sets the baseline individually for each well Once you select the wells for analysis check the baseline settings in these wells Open the Baseline Thresholds window Figure 75 to change the default baseline for selected wells To open this window 1 Select one fluorophore in the fluorophore selector in the Quantitation tab Figure 72 by clicking the boxes next to th...

Page 84: ...ta as baseline subtracted traces and the software smoothes the baseline subtracted curve using a centered mean filter This process is performed so that each C t is left invariant Well Selectors Click the wells in the well selector to show or to hide the data in the charts or spreadsheets throughout the Data Analysis window To hide one well highlight and click the individual well To show that well ...

Page 85: ...nalysis temporarily follow these instructions RIGHT CLICK OPTION 1 Right click on the well in the well selector on a fluorescence trace or on a point plotted on the standard curve Table 21 Right click menu items in the well selectors Item Function Copy Copy the content of the well to a clipboard including Sample Type and optional Replicate Copy as Image Copy the well selector view as an image Prin...

Page 86: ...ck menu to reinclude the well PLATE EDITOR OPTIONS 1 Click the View Edit Plate button on the toolbar in the Data Analysis window 2 Select one or more wells in the well selector view 3 Click Exclude Wells in Analysis Figure 78 to exclude the selected wells This checkbox is at the bottom of the Plate Editor controls on the right side of the window Figure 78 Exclude Wells in Analysis checkbox at bott...

Page 87: ...e chart and selecting Set Scale to Default from the right click menu Common Right Click Menu Items for Charts Right click menu items are available on all charts Some of the available items are present for all charts and these items can be used to change how the data are displayed or to easily export the data from a chart Table 22 Table 22 Right click menu items for charts Item Function Copy Copy t...

Page 88: ...ick OK to sort the data or click Cancel to stop sorting Highlight the data on the associated charts and well selector by holding the mouse pointer over a cell If you click in the cell you can copy the contents to paste into another software program Common Right Click Menu Items for Spreadsheets Right click any spreadsheet view to select the items shown in Table 23 Table 23 Right click menu items f...

Page 89: ...Data Analysis Overview 80 ...

Page 90: ...ng the baseline settings for individual wells and the threshold settings The Quantitation tab shows data in these four views Amplification chart Shows the relative fluorescence units RFUs for each well at every cycle Each trace in the chart represents data from a single fluorophore in one well Standard curve This graph is only shown if the experiment includes wells designated as Sample Type Standa...

Page 91: ...orophore name to show or hide the fluorophore data throughout the data analysis window Figure 81 Fluorophore selector with FAM selected Trace Styles Window Open the Trace Styles window Figure 82 to adjust the appearance of traces in the amplification and melt curve charts in the Quantitation and Melt Curve tabs To open this window follow these steps 1 Select only one fluorophore in the fluorophore...

Page 92: ...x in the Color column to select a color for the wells Select a symbol from the pull down menu in the Symbol column Click Show Contents to show the sample types in each well or click Show Symbols to show the selected Symbols in each well Click Remove Symbols to remove all the added symbols from all wells Click Restore Default Colors to return to the default trace colors Log Scale Option Click the L...

Page 93: ... that you are doubling your target with each cycle Coefficient of determination R2 written as R 2 Use this statistic to determine how correctly the line describes the data goodness of fit Chart Right Click Menu Options In addition to the common right click menu options to copy print and export charts Table 24 lists the menu options available only on the Amplification chart Table 24 Amplification c...

Page 94: ...t option Results Spreadsheet Select a Results spreadsheet Figure 85 to see data for each well in the plate Figure 85 Quantitation Data tab with Results spreadsheet selected NOTE All Std Dev standard deviation calculations apply to the replicate groups assigned in the wells in the Plate Editor window The calculations average the C t value for each well in the replicate group The Results spreadsheet...

Page 95: ...the replicate group C t Std Dev Standard deviation of the threshold cycle for the replicate group Starting Quantity SQ Estimate of the starting quantity of the target Log Starting Quantity Log of the starting quantity SQ Mean Mean of the starting quantity SQ Std Dev Standard deviation of the starting quantity Set Point Temperature of sample in the well for a gradient step Sample Note One round of ...

Page 96: ...lexes The software plots the RFU data collected during a melt curve as a function of temperature To analyze melt peak data the software assigns a beginning and ending temperature to each peak by moving the threshold bar The floor of the peak area is specified by the position of the melt threshold bar A valid peak must have a minimum height relative to the distance between the threshold bar and the...

Page 97: ... protocol The list shows more than one step if the protocol includes plate read camera icon in two or more melt curve steps Select wells in the well selector to focus on subsets of the data Select a well group page 73 to view and analyze a subset of the wells in the plate Select each well group by name in the Well Group pull down menu in the toolbar Melt Curve tab Spreadsheet Table 29 shows the ty...

Page 98: ...on plot for each well in the plate Melt Peaks Spreadsheet Select the Melt Peaks spreadsheet Figure 89 to view melt curve data Figure 89 Melt Peaks spreadsheet in Melt Curve Data tab The Melt Peaks spreadsheet Figure 89 includes the type of information shown in Table 30 Table 30 Melt Peaks spreadsheet content Information Description Well Well position in the plate Fluor Fluorophore detected Content...

Page 99: ... Curve tab The Plate spreadsheet includes the types of information shown in Table 31 RFU Spreadsheet Select the RFU spreadsheet to view the fluorescence for each well at each cycle acquired during the melt curve Figure 91 Figure 91 RFU spreadsheet in Melt Curve Data tab Table 31 Plate spreadsheet content Information Description Content A combination of Sample Type required and Replicate optional S...

Page 100: ... must contain negative controls or the software cannot make the call Run one of these two types of protocols Run a Quantitation protocol Set up a standard protocol After running the experiment open the Data Analysis window adjust the data analysis settings in the Quantitation tab and then click the End Point tab to pick an end point cycle Run an End Point Only protocol Load the End Point Only prot...

Page 101: ... the data Highest RFU value Highest RFU value in the data Negative Control Average Average RFU for the wells that contain negative controls Cut Off Value Calculated by adding the tolerance RFU or Percentage of Range listed in the Settings and the average of the negative controls Samples with RFUs that are greater than the cut off value will be called Positive To adjust the cut off value change the...

Page 102: ...1 or Control 2 NOTE The data for allelic discrimination must come from multiplex experiments with at least two fluorophores Each fluorophore identifies one allele in all samples Allelic discrimination analysis requires the following minimal well contents Two fluorophores in each well except the wells that contain positive controls can contain only one fluorophore One fluorophore that is common to ...

Page 103: ...et the threshold lines for discriminating the alleles Adjust the position of the threshold bars by clicking and dragging them and the software automatically adjusts the calculations to make new genotype assignments If the experiment contains three controls in the plate then the position of the threshold bars is based on the mean and standard deviation of the RFU or C t of the controls If the numbe...

Page 104: ... effective way to present RFU data The calculations for normalized RFU follows the formulas presented in Livak et al 1995 Where A1 represents RFU for Allele 1 A2 represents RFU for Allele 2 represents the mean RFU NTCA1 A2 represents the sum of RFUs for the NTC sample of Allele 1 and Allele 2 Allelic Discrimination tab Spreadsheet The Allelic Discrimination spreadsheet at the top right side of the...

Page 105: ...Rule column Well selector Selects the wells with the fluorescence data you want to show Rule Description Shows the selected QC rule and highlights wells that fail the rule Figure 95 QC tab layout Run Information Tab The Run Information tab Figure 96 shows the protocol and other information about the run for each experiment Open this tab for the following options View the protocol Enter and edit th...

Page 106: ...o cut copy paste delete or select the text Reports for Data Files The Report window Figure 97 shows information about the current data file in the Data Analysis window To open a report select Tools Reports or click the Reports button on the toolbar in the Data Analysis window The Report window shows these three sections Menu and toolbar Select options to format save and print the report or templat...

Page 107: ...ort options list to change whole categories or individual options within a category NOTE The data that appear in the report are dependent on the current selections within the tabs of the Data Analysis window For example a quantitation experiment might not contain a standard curve and therefore those data do not appear in the Data Analysis window or in the data report 4 Click the Update Report butt...

Page 108: ...ep number when data were collected the analysis mode and the baseline subtraction method Amplification Chart Copy of the amplification chart for experiments that include quantitation data Standard Curve Chart Copy of the standard curve chart Data Spreadsheet listing the data in each well Gene Expression Analysis Settings Includes the analysis mode chart data scaling option and chart error Chart Co...

Page 109: ...ings Includes fluorophore end cycles to average mode lowest RFU value highest RFU value and cut off value Data Spreadsheet listing the data in each well Table 36 Data analysis report categories in the options list continued Category Option Description ...

Page 110: ...hey should not be regulated in the biological system being studied Open the Gene Expression tab to evaluate relative differences between PCR reactions in two or more wells For example you can evaluate relative numbers of viral genomes or relative number of transfected sequences in a PCR reaction The most common application for gene expression study is the comparison of cDNA concentration in more t...

Page 111: ...ment Settings window page 48 The requirements for Gene Expression setup in the Plate Editor depend on whether reaction contents are singleplex PCR with one fluorophore in the reactions or multiplex PCR with more than one fluorophore in the reactions Figure 98 shows an example of the minimum contents of the wells for a singleplex gene expression experiment Figure 98 Example of well contents in a si...

Page 112: ...ssion ΔΔC t analysis follow these steps 1 Open a data file pcrd extension 2 Review the data in the Quantitation tab of the Data Analysis window Make adjustments to the data such as changing the threshold and the Analysis Mode 3 Click the Gene Expression tab 4 Choose a control in the Samples tab of the Experiment Settings window If a control is assigned the software normalizes the relative quantiti...

Page 113: ... your analysis method adjust the data you view in the Gene Expression tab by changing the settings options to the right of the chart GRAPH DATA Graph data options allow you to present the data in the graph with one of these two options Relative to control Graph the data with the axis scaled from 0 to 1 If you assign a control in your experiment select this option to quickly visualize upregulation ...

Page 114: ...an 1 reference gene is used The software calculates two quality parameters for the reference genes Coefficient of Variation CV of normalized reference gene relative quantities Lower CV values denotes higher stability M value A measure of the reference gene expression stability Right Click Menu Options for Gene Expression Graph Right click on the Gene Expression graph to select the items shown in T...

Page 115: ...xpression SEM or SD Corrected value calculation for Standard Error of the Mean SEM or Standard Deviation SD of the relative expression depending on the selected option Mean C t Mean of the threshold cycle C t SEM or SD Standard Error of the Mean or Standard Deviation of the threshold cycle depending on the selected option Table 39 Information in Gene Expression spreadsheet with Show Details select...

Page 116: ... as a control sample for gene expression data analysis by clicking the box in the Control column next to the name for that sample Sample Name Grouping Option Loading Collection Names in the wells enables samples to be analyzed in one of four configurations defined by the Sample Name Grouping Option These options are available from the pull down menu in the Experiment Settings tab Target vs Sample ...

Page 117: ...ncy is selected Figure 102 Targets tab in Experiment Settings window with Analysis Settings selected To adjust the settings for a sample in the Samples tab Click a color in the Color column to change the color of the samples graphed in the Gene Expression chart Click a box in the Show Graph column to show the sample in the Gene Expression chart using a color that is selected in the Color column Fi...

Page 118: ... a given target gene and then uses a multitiered algorithm to determine the dominant inter run calibrator within all the data The algorithm for finding the dominant inter run calibrator includes the following hierarchy 1 Set the dominant calibrator to the target with the highest number of common replicate groups in a given pair wise comparison 2 If any target has the same number of common replicat...

Page 119: ... each experiment before you add them to a Gene Study Select the well group you want to include in the Gene Study The Study Setup tab Figure 104 shows a list of all the experiments in the Gene Study Add experiments Click the Add Data Files button to select a file from a browser window To quickly add experiments to a Gene Study drag the data files pcrd extension to the Gene Study window TIP In order...

Page 120: ... Error Select the multiplier for standard deviation bars in the graph including 1 2 or 3 Experiment Settings button Choose the show options for targets and samples in the Experiment Settings window Show Details check box Click Show Details to add more columns of data to the chart Table 40 Study Setup tab in Gene Study window Column Title Description File Name Name of the experiment data file pcrd ...

Page 121: ...d gene selected in the Experiment Settings window Sample Sample Name selected in the Experiment Settings window Ctrl Control sample when the sample name is selected as a control in the Experiment Settings window Expression Normalized Gene Expression ΔΔC t or Relative Quantity ΔC t depending on the selected mode Expression SEM or SD Standard Error of the Mean or Standard Deviation depending on the ...

Page 122: ...le Relative Quantity Calculated relative quantity of samples Relative Quantity SD Standard deviation of the relative quantity calculation Corrected Relative Quantity SD Calculated standard deviation of the corrected relative quantity Unscaled Expression Calculated unscaled expression Unscaled Expression SD Calculated standard deviation unscaled expression Corrected Unscaled Expression SD Corrected...

Page 123: ...orts To create a template select Template Save or Save As and save the current report as a template Frequently Asked Questions The following list is a series of questions Q and answers A about gene expression analysis in CFX Manager software Q Why should I normalize my data A Relative quantity data that is not normalized by some means is difficult to interpret Imagine the case where you load 1 μg ...

Page 124: ...48 to add names to the Targets or Samples tabs where you can also enter or remove the full names from the lists Alternatively permanently add long lists of names to the Libraries for target and sample names in the Plate tab in the User Preferences window page 125 These names appear on the axis in various chart views including gene expression Q How do I determine efficiencies A Typically the effici...

Page 125: ...Is Selected When a control sample control is assigned then the relative quantity RQ for any sample GOI with a gene of interest is calculated with this formula Where E Efficiency of primer and probe set This efficiency is calculated with the formula Efficiency 0 01 1 where 100 efficiency 2 CT control Average C t for the control sample CT sample Average C t for any samples with a GOI GOI Gene of int...

Page 126: ...lative Quantity RQ calculation Where RQ Relative Quantity of a sample Ref Reference target in an experiment that includes one or more reference targets in each sample GOI Gene of interest one target Provided that reference targets do not change their expression level in your biological system the calculation of normalized expression will account for loading differences or variations in cell number...

Page 127: ...rmula Where NE Normalized Expression RQ Relative quantity of a sample SD standard deviation GOI Gene of interest one target Normalized Expression Scaled to Highest Expression Level When the experiment does not include controls scale the normalized expression NE for each target gene by dividing the expression level of each sample by the highest level of expression in all the samples The software se...

Page 128: ...hich scaling option you choose NOTE When a control sample is assigned you do not need to perform this rescaling function on the standard deviation The formula for this calculation is shown here Where NE Normalized expression SD Standard deviation GOI Gene of interest target MAX Highest expression level MIN Lowest expression level Corrected Values Formulas A difference between corrected values and ...

Page 129: ...ized expression RQ Relative quantity The standard error for normalized gene of interest GOI formula is shown here Where SE Standard error GOI Gene of interest one target NF Normalization factor n Number of reference targets SE NFn NFn SE RQsample Ref 1 n SE RQsample Ref 1 2 SE RQsample Ref 2 n SE RQsample Ref 2 2 SE RQsample Ref n n SE RQsample Ref n 2 SE GOIn GOIn SE NFn NFn 2 SE GOI GOI 2 ...

Page 130: ...name displayed CFX Manager software manages who logs in to the software through the Login dialog box Figure 109 When you start the software the Login dialog box opens automatically if there are two or more users listed in the User Administration window Figure 109 Login dialog box Log in to the software or switch users by following these steps 1 Open the Login dialog box if it is not already open b...

Page 131: ...New Password and the Confirm New Password boxes 4 Click OK to confirm the change Figure 110 The Change Password window User Preferences Window CFX Manager software tracks the preferences of each user that logs in to the software To change user preferences open the User Preferences window using one of these methods Click the User Preferences button in the main software window toolbar Select User Us...

Page 132: ...this is usually 25 Use SSL Whether to use Secure Sockets Layer Some SMTP servers require this to be used others require that it not be used Use Default From Address This can usually be left in the default checked state However some SMTP servers require all sent email to have a from address that is from a certain domain i e name YourCompany com If that is the case this checkbox must be unchecked an...

Page 133: ...e the beginning text of the file name for data files The default setting instructs the software to create a file name that starts with the User user name of the user who is currently logged on to software Date file creation date and Instrument Name instrument serial number or name Figure 113 Files Preferences tab in the User Preferences window TIP Click the button to the right of each box to open ...

Page 134: ...the units used to describe the concentration of the starting template for wells that contain standards The software uses these units to create a standard curve in the Data Analysis Quantitation tab Scientific Notation Select scientific notation to view concentration units in that notation Scan Mode Select a default scan mode to set the number of channels to scan during a run Fluorophores Click che...

Page 135: ...w Data Analysis Tab Select the Data Analysis Tab in the User Preferences window to change the default settings for data that appear in the Data Analysis window Figure 116 Data Analysis tab in the User Preferences window For the quantification data select the following settings ...

Page 136: ...e control sample For the end point data select the following settings Select the number of end cycles to average when calculating the end point calculations PCR Enter a number of cycles for PCR to average the end cycles for quantitation data default is 5 End Point Only Run Enter a number of cycles for End Point Only Run to average the end cycles for end point data default is 2 Gene Expression Tab ...

Page 137: ...ror bars The default is 1 Change the multiplier to either 2 or 3 QC Tab Select the QC tab in the User Preferences window to specify QC rules to apply to data in Data Analysis Module The software validates the data against the enabled tests and the assigned values page 128 NOTE Wells that fail a QC parameter can easily be excluded from analysis in the QC module of the Data Analysis Window using the...

Page 138: ...ser Administration window to manage users and user rights Manage Users Add or remove Users and assign each user a Role Manage Rights Change rights for user roles Principal Operator or Guest NOTE Only users who are Administrators can edit this window Other users can only view it To assign a role to each user select from the list of roles in the User Administration window Figure 119 In this example ...

Page 139: ...e rights for each role Principal By default each Principal has all rights Operator By default each Operator has all rights except skipping cycles and creating a Gene Study Guest By default each Guest has no additional rights and can only read files To specify the rights for each role follow these steps Only a software Administrator can change the rights for any role 1 In the Manage Rights pane cli...

Page 140: ...led plates only Table 44 The CFX96 system or the CFX384 system also include a channel dedicated for FRET chemistry this channel does not require calibration for specific dyes To open the Calibration Wizard to calibrate the CFX96 or CFX384 real time PCR system 1 Select an instrument in the Detected Instruments pane 2 Select Tool Calibration Wizard to open the window and calibrate new dye and plate ...

Page 141: ...eat steps 1 6 to add each fluorophore you plan to calibrate for the plate 6 When you finish adding fluorophores click View Plate to open the Dye Plate Display Use this window as a guide for loading dyes into the plate 7 Begin preparing a 96 or 384 well plate for dye calibration by pipetting dye solution into each well following the pattern shown in the Pure Dye Plate Display For each fluorophore f...

Page 142: ...that are in the inner lid Figure 121 on page 134 Clean the outer lid and C1000 base on a regular schedule for details see C1000 thermal cycler instruction manual Cleaning the Optical Reaction Module The block of the optical reaction module should be cleaned along with the C1000 thermal cycler base on a regular schedule to remove any debris or dirt that might interfere with proper function Clean as...

Page 143: ... sample heating and cooling NOTE Never use cleaning solutions that are corrosive to aluminum such as bleach or abrasive cleansers Use of oil in the wells is not recommended If oil is used the wells must be cleaned thoroughly and often Remove the oil when it is discolored or contains dirt Use a solution of 95 ethanol to clean oil Do not allow oil to build up in the block Clean the wells in the bloc...

Page 144: ...t when the problem started happening CFX Manager software tracks information about the state of an instrument during a run in the Application Log Figure 122 Use these logs to track events that occur on instruments and in the software and for troubleshooting To open the Application log in the main software window select View Application Log Figure 122 Example of an Event Log file Software Help Tool...

Page 145: ...izard to install the software and then click Finish Installing the Drivers Manually If needed install the drivers manually by following these instructions 1 Insert the software CD If the CD is not available then locate the drivers folder in the file path C Program Files Bio Rad Drivers on your hard drive 2 Click the Drivers button software installation screen Figure 124 3 Click the BaseUnit folder...

Page 146: ...s not shut down by the computer When the computer and software start up again then the protocol continues If you want to open a locked motorized lid on a reaction module to remove your samples during a power failure follow these steps to remove the locking plate 1 Remove the reaction module from the C1000 chassis by pushing down on the locking bar of the C1000 2 Position the module on the front of...

Page 147: ...ion Module 185 1048R C1000 Thermal Cycler With Dual 48 48 Fast Reaction Module 185 1384R C1000 Thermal Cycler With 384 Well Reaction Module 185 2096R S1000 Thermal Cycler With 96 Well Fast Reaction Module 185 2048R S1000 Thermal Cycler With Dual 48 48 Fast Reaction Module 185 2384R S1000 Thermal Cycler With 384 Well Reaction Module Software and Accessories 184 5000 CFX Manager Software 184 5001 CF...

Page 148: ...tice 1999 University of Chicago All rights reserved Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met 1 Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer 2 Redistributions in binary form must reproduce the above copyright notice this lis...

Page 149: ...Resources 140 ...

Page 150: ...ng temperature Protocol AutoWriter 38 39 Auto Efficiency 50 108 B Back panel 3 Balance Cut microplates 10 How to 10 Tube strips 10 Baseline Window 74 Baseline Subtracted Curve Fit mode 75 Baseline Subtracted mode 75 Baseline Threshold window 74 Beep 37 Bio Rad Laboratories Contact ii Resources ii Web site ii Block 3 Mode 37 Button 27 Clear Replicate 48 Clear Wells 48 Close lid 3 26 Delete step 37 ...

Page 151: ... adjusting 78 Clearing wells 78 Data analysis 93 End Point 91 Excluding wells 76 Formula 115 Gene Expression 101 Melt Curve 87 Menu bar 71 Plate content 69 Toolbar 70 Well contents 69 Well groups 50 Well selector 75 Data file Report 97 Data report Creating 98 Delete Step button 37 Deleting a step 37 Detected Instruments 12 Dilution Series 48 E Email function on the C1000 62 Email Setup in CFX Mana...

Page 152: ...paring data 110 Report 113 Show Details 113 Study Analysis tab 109 111 Study Setup tab 110 Glossary Software 135 GOI Normalization factor 117 Normalized expression 117 118 119 Relative quantity 116 SD Normalized expression 119 GOTO Adding repeats 27 Inserting 35 Gradient Inserting 34 Step 34 Gradient Calculator 32 Guides Quick Guides 11 Software Help 11 H Heated plate 3 Help tools 135 Software 135...

Page 153: ...un 27 PCR Supermixes 138 Plastic Consumables 9 Plate Contents of wells 46 Files 20 Melt Curve Data 90 Size in Plate Editor 43 Spreadsheet 51 90 Spreadsheet in Plate Editor 42 Type 43 Well contents 46 Plate Editor Clear Replicate 48 Clear Wells button 48 Clearing wells 78 Concentration 47 Dilution Series 48 Excluding wells 76 Experiment Settings 48 Export plate spreadsheet 52 Import plate spreadshe...

Page 154: ... Replicate Group 47 Replicate Series 47 Report Data file 97 Gene Study 113 Requirements Operation 1 Resume button 27 Resuming a run 27 Reverse transcription Reagents 138 RFU Allelic Discrimination 93 96 End Point tab 91 92 Melt Curve tab 87 Right click Gene expression graph 105 Selected Block s list 25 RT_Data Folder 61 Run Adding repeats 27 Cancelling 27 Flash block 27 Pausing 27 Skipping a step ...

Page 155: ...y 116 Standard error 119 Formula 119 Normalized expression formula 119 Startup Wizard 11 15 Step Temperature 33 Stop button 27 Stopping a run 27 Study Analysis tab 109 111 Study Setup tab 109 110 Support Bio Rad representatives ii Technical ii Technical Notes ii SYBR FAM only 45 60 T Tab End Point 92 Gene Expression 102 Melt Curve Data 89 Quantitation 70 72 Real Time Status 27 Run Information 96 S...

Page 156: ...Well Concentration 46 Contents 46 Fluorophores 46 Groups 44 Notes 46 48 Sample Name 46 Sample Type 46 Target Name 46 Well groups 50 Creating 50 Data analysis 50 Standard Curve 50 Well Groups Manager 44 Well Notes 48 Well selector Data analysis 75 Window Baseline 74 Gene Study 109 Plate Editor 42 Protocol AutoWriter 37 Protocol Editor 31 Run Details 25 Threshold 74 Well Groups Manager 44 Writing co...

Page 157: ...Index 148 ...

Page 158: ... 01 47 95 69 65 Germany 089 318 84 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South ...

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