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1. Figure 67 Example of well contents in a multiplex gene expression experiment Gene Expression Tab The Gene Expression tab in the Data Analysis window shows the relative expression of targets in these two views e Gene Expression chart Shows the real time PCR data as normalized expression AAC t or relative quantity AC t e Spreadsheet Shows a spreadsheet of the gene expression data TIP Right click any chart or spreadsheet for options Click the View Edit Plate button to open the Plate Editor and change well contents in the plate 80 CFX96 and CFX96 Deep Well Systems Instruction Manual Va Quantitation Me Quantitation Data alll Gene Expression coe End Point Allelic Discrimination Kh QC a Fun Information c 2 n n a x lt LL Dd O LL D Ke N w E a O Mode Normalized expression AAC t v Gene Expression j 2 Graph Data 15 Axis g Sample v Y Axis 11 2 Log 2 v 3 Scaling Mo N M NMN N u on Error Type Standard Deviation v Chart Error Bar Multiplier 1 Std Devs el Experiment Settings AT arget Stability Value Sample C Show Details M Corrected Target 4 Sample 9 Ctl Expression Q Sac Expression MeanCit gt Cit SD 9 SD 2Hr 26 22167 3 41157 4 11364 24 31 0 11801 3Hr 316 83363 43 01324 65 81114 21 07 0 14077 4Hr 4911 76997 538 54542 1251 38823 17 68 0 12201 5Hr 70738 16818 6685 17308 22451 26096 14 28 0
2. Figure 69 Experiment Settings window with Targets tab selected 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 by clicking the Select to Remove Name box for that row and then clicking the Remove checked item s button Select the target as a reference for gene expression data analysis by clicking the box in the Reference column next to the Name for that target Select the sample 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 Only the well sample name is used in the gene expression calculations Target vs Collection Only the well collection name is used in the calculations Target vs Sample_Collection The sample name and collection name are combined to make a single name that is used in the calculations Target vs Collection_Sample The collection name and sample name are combined to make a single name that is used in the calculations Show Analysis Settings in Experiment Settings Click the Show Analysis Settings box in the Experiment Set
3. In the Calibrate New or Existing Fluorophores pane select the fluorophore you want to calibrate from the pull down list If the fluorophore name is not included in the list type the name in the box to add it to the list Select the Plate Type If the plate type is not included in the list type the name in the box to add it to the list Select a Channel for the fluorophore Click Add to Plate to add the fluorophore To clear the plate click Clear Plate to remove all the fluorophores Optional Repeat steps 1 6 to add each fluorophore you plan to calibrate for the plate 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 Prepare a 96 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 fill 4 wells with 50 ul 96 well plate of 300 nM dye solution Notice that at least half the plate contains blank wells Seal the plate using the sealing method you will use in your experiment Place the calibration plate in the block and close the lid Then click Calibrate and click OK to confirm that the plate is in the block 10 When the CFX Manager software completes the calibration run a dialog box appears Click Yes to finish calibration and open the Dye Calibration Viewer 11 Click OK to close the window 103 Resources Instrument
4. e Place the 0 2 ml microplate or tube strips with sealed lids in the block Check that the tubes are completely sealed to prevent leakage For optimal results load sample volumes of 10 25 ul for the CFX96 system and 10 125 ul for the CFX96 Deep Well system NOTE For accurate data analysis check that the orientation of reactions in the block is exactly the same as the orientation of the well contents in the software Plate tab If needed edit the well contents before during or after the run System Installation WARNING When running the CFX system always balance the tube strips or cut microplates in the wells Figure 8 For example if you run one tube strip on the left side of the block run an empty tube strip with caps on the right side of the block to balance the pressure applied by the heated lid Failure to balance the pressure can result in sample evaporation and failed runs Filled tube Tube strip for strip balance Figure 8 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 Shutting Down the System To shut down the CFX system follow these suggestions e After arun click the open lid button on the front of the CFX system to access the samples loaded in the thermal cycler block
5. 1 Enter a User Name for the new software user 2 Select a user Role These roles restrict the rights of each user The default is Principal 3 Optional Enter a Full Name and Password for the new software user 4 Click OK to open a dialog box and confirm that you want to close the window 5 Click Yes to close the dialog box and window 97 Users and Preferences 98 To remove a software user follow these steps 1 In the Manage Users pane click the box in the Delete list for each software user you want to remove Click OK to open a dialog box and confirm that you want to close the window Click Yes to close the dialog box and window NOTE The list of software users must always include one Administrator Assign Rights for User Roles The User Administration window provides access to user roles and rights The software includes these four roles Administrator required Each Administrator has all rights and you cannot change those rights The Administrator can also add and remove software users and change the 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 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 k 2 3 In the Manage R
6. CF X96 and CFX96 Deep Well Real Time PCR Detection Systems Instruction Manual 184 8097 VD 185 4095 IVD 184 4095 IVD Manual revision October 2014 Software revision 1 6 CE Copyright 2014 Bio Rad Laboratories Inc Reproduction in any form either print or electronic is prohibited without written permission of Bio Rad Laboratories Inc Excel Microsoft PowerPoint Windows Windows 7 and Windows 8 are trademarks of Microsoft Corporation Adobe and Reader are trademarks of Adobe Systems Incorporated Cy is a trademark of GE Healthcare group companies CAL Fluor and Quasar are trademarks of Biosearch Technologies Inc SYBR and Texas Red are trademarks of Life Technologies Corporation Bio Rad Laboratories Inc is licensed by Life Technologies Corporation to sell reagents containing SYBR Green for use in real time PCR for research purposes only EvaGreen is a trademark of Biotium Inc FAM ROX and VIC are trademarks of Applera Corporation Other brands or product names are trademarks of their respective holders NOTICE REGARDING BIO RAD THERMAL CYCLERS AND REAL TIME SYSTEMS This product is covered by one or more of the following U S patents or their foreign counterparts owned by Eppendorf AG U S Patent Numbers 6 767 512 and 7 074 367 Bio Rad s Hard Shell plates are covered by one or more of the following U S patents patents pending or their foreign counterparts owned by Eppendorf AG US Patent Nos 7 347
7. These wells contain a loaded Unk unknown sample type The data from these wells appear in the Data Analysis window e Unselected loaded wells light gray These wells contain loaded Std and Pos sample types The data from unselected wells do not appear in the Data Analysis window e Empty wells dark gray These wells were not loaded in the Plate Editor window ownemoeo 7 gt Figure 50 Three well colors appear in a well selector 60 CFX96 and CFX96 Deep Well Systems Instruction Manual Temporarily Exclude Wells from Analysis 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 2 Choose Exclude Well XX from Analysis from the menu options Amplification an l ig Exclude Well B3 from Analysis Copy m a 2 Save Image As Page Setup Print Show Paint Values Set Scale to Default Chart Options Figure 51 Right click to exclude a well from analysis 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 52 to exclude the selected wells This checkbox is at the bottom of the Plate Editor controls on the right side of the window sl Experiment Settings Clear Replicate Bi Clear Wells Hh xclude Wells in Analysis i Figure 52
8. 00 eee 93 Ser AGiministraQuOM schvw ke dere amp n a a bone ek aoe eh et eee ee at ne 97 Chapter 11 Resources 6 cecoe ee cee a Se Se eee ee eae eee 99 LIMS IMIS OratiOn chc pie ete hee at Be ie a ete eh ee Beh eee ea 99 calbratom Wizard zerei iir a a A dew ie dee ds E a A 102 Instrument Maintenance 00 eee ees 104 POPICAIOW DOO wickets pace ees cide oa thew eae eee Ronee ee eA 106 IFOUDICSMOOUNG r cde soi date dee ee Wie eh oh eee ee ee 106 FICICICNCCS i cag sos EDF he Sak Hae AS Oe Ne aes oO ea eS ee 108 viii CFX96 and CFX96 Deep Well Systems Instruction Manual 1 System Installation Read this chapter for information about setting up the CFX system e Unpacking the optical reaction module page 1 e System requirements page 1 e System overview page 2 e Setting up the system page 3 e Installing CFX Manager software page 5 e Software files page 7 e Running experiments page 7 Unpacking the Optical Reaction Module The optical reaction module shipment includes these components e Optical reaction module e USB cable e CFX Manager software installation CD e Instruction manual 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 CFX system use the following power sources and cables e Input power 100 240 VAC 50 60 Hz e Indoor use Ambient temperature 15 31 C Relative humidity
9. A gradient step cannot include an increment or ramp rate change Step Options Step 1 Gradient Plate Read A 100 0 Temperature ogg C Gradient Increment Cicycle Ramp Rate Cisec Time 3 sec cycle Extend secicycle H 90 0 Figure 23 Step option for a gradient 29 Protocols The Step Options window lists the following options you can add or remove from steps e Plate Read Check the box to include a plate read e Temperature Enter a target temperature for the selected step e Gradient Enter a gradient range for the step e Increment Enter a temperature to increment the selected step the increment amount is added to the target temperature with each cycle e Ramp Rate Enter a rate for the selected step the range depends on the block size e Time Enter a hold time for the selected step e Extend Enter a time to extend the selected step The extend amount is added to the hold time with each cycle e Beep Check the box to include a beep at the end of the step TIP When you enter a number that is outside the option range the software changes the number to the closest entry within the range Delete Step Button To delete a step in the protocol 1 Select a step in the graphic or text view 2 Click the Delete Step button to delete the selected step WARNING You cannot undo this function Temperature Control Mode The instrument uses one of two temperature control modes to determine when
10. Before beginning the run the software verifies that the fluorophores you specified in the plate are calibrated on that instrument You cannot run a plate if it includes fluorophores that have not been calibrated on that instrument Well Loading Controls A plate file contains information about the contents of each well loaded with sample for an experiment After the run the software links the well contents to the fluorescence data collected during the protocol and applies the appropriate analysis in the Data Analysis window For example wells loaded with standard sample type are used to generate a standard curve Consider the following guidelines for well contents e Target Name One or more targets of interest genes or sequences in each loaded well Each target is assigned to one fluorophore e Sample Name One identifier or condition that corresponds to the sample in each loaded well such as O hr 1 hr or 2 hr 36 CFX96 and CFX96 Deep Well Systems Instruction Manual 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 14 Table 14 Options for loading the plate and wells in the Plate Editor Option Sample Type U nknown Unknown Load FAM Positive Control Negative Control HE
11. Est Run Time 01 09 00 F 1 0 C f 300 mi Insert Step 2 950 C for010 3 550 C for 0 30 Insert Gradient PlateRead _ 4 GOTO 2 39 more times END ce Add Plate Read to Step Figure 20 Protocol Editor window with buttons for editing protocols Protocol Editor Menu Bar The menu bar in the Protocol Editor window provides the menu items listed in Table 11 Table 11 Protocol Editor menu bar Menu Item Function File Save the current protocol Save As Save the current protocol with a new name or in a new location Close the Protocol Editor Settings Lid Settings Open the Lid Settings window to change or to set the Lid Temperature Tools Gradient Select the block type for a gradient step Calculator Run time Select the instrument and scan mode to be used for Calculator calculating the estimated run time in the Experiment Setup window 26 CFX96 and CFX96 Deep Well Systems Instruction Manual The toolbar in the Protocol Editor window provides quick access for important functions Table 12 lists the function of the Protocol Editor toolbar buttons Table 12 Protocol Editor toolbar buttons Toolbar Button and Menus Function Save Save the current protocol file Print Print the selected window Insert Step Select After or Before to insert a step relative to the currently highlighted step Insert Step After NI After Sample Volume Enter a sample volume in ul between 0 and Sample Volume 5 u
12. Exclude Wells in Analysis checkbox at bottom of the pane 4 Excluded wells are marked with an asterisk in the Plate Editor window Alternatively to permanently remove wells from analysis clear the contents from wells in the Plate Editor by clicking the Clear Wells button WARNING You will have to reenter any well content that is cleared 61 Data Analysis Overview Charts Each chart in the Data Analysis window displays the data in a different graph and includes options for adjusting the data To magnify an area of the chart select an area by clicking and dragging the mouse The software resizes the chart and centers it on the selected area 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 18 Table 18 Right click menu items for charts ltem Function Copy Copy the chart into the clipboard Save Image As Save the chart image in the selected image file type Select from these formats PNG default GIF JPG TIF or BMP Page Setup Preview and select page setup for printing Print Print the chart Show Point Values Show the point values when the mouse moves over a point on the chart Set Scale to Default Return to default chart view after magnifying the chart Chart Options Open the Chart Options
13. Figure 56 Standard Curve chart The Standard Curve chart displays the following information Name for each curve the fluorophore name Color of each fluorophore Reaction efficiency E Use this statistic to optimize a multiplex reaction and equalize the data for a standard curve NOTE The reaction efficiency describes how much of your target is being produced with each cycle in the protocol An efficiency of 100 means that you are doubling your target with each cycle Coefficient of determination R 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 20 lists the menu options available only on the Amplification chart Table 20 Amplification chart specific right click menu options Menu Option Function Show Threshold Values Display the threshold value for each amplification curve on the chart Trace Styles Open the Trace Styles window to change trace styles that appear on the Quantitation and Melt Curve tabs Baseline Thresholds Open the Baseline Thresholds window to change baseline or thresholds of each fluorophore changes appear in Amplification chart in Quantitation tab 67 Data Analysis Windows Quantitation Data Tab 68 The Quantitation Data tab shows spreadsheets that describe the quantitation data collected in each well
14. Select one of the three options to show the data in different formats e Results Displays a spreadsheet view of the data e Plate Displays a view of the data in each well as a plate map e RFU Choose this spreadsheet to show the RFU quantities in each well for each cycle TIP Right click any spreadsheet for options including the sort option Results Spreadsheet Select a Results spreadsheet Figure 57 to see data for each well in the plate Va Quantitation F Quantitation Data alll Gene Expression oe End Point Allelic Discrimination Kh Qc bY Run Information Results vy Step Number 3 j i Threshold i A Starting Quantity Log Starting i Well Fluor Content Target Sample 7 Cycle Ct gt Cit Mean Cft Std Dey 6 50 X SQ Mean Quantity A02 FAM Std 1 NGK 5Hr 14 39 14 28 0 081 1 000E 06 6 000 1 00E 06 B02 FAM Std 1 NGK 5Hr 14 24 14 28 0 081 1 000E 06 6 000 1 00E 06 c02 FAM Std 1 NGK 5Hr 14 25 14 28 0 081 1 000E 06 6 000 1 00E 06 D02 FAM Std 1 NGK 5Hr 14 22 14 28 0 081 1 000E 06 6 000 1 00E 06 A02 HEX Std 1 ACT 5Hr 13 27 13 13 0 101 1 000E 06 6 000 1 00E 06 Figure 57 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 includes the type of information
15. attachments by checking the Test Attachment box and setting the Attachment Size in MB with up to 5 megabytes MB or more Files Tab Select the Files tab to list the default locations for opening and saving files Click the button to the right of each box to open a browser window and locate a folder e Default Folder for File Creation Select a default folder where you want to save new files Select a location for each file type Protocol Plate Data or Gene Study file 93 Users and Preferences 94 e File Selection for Experiment Setup Select the default protocol and plate files that appear when you open the Experiment Setup window e Data File Prefix Define the beginning text of the file name for data files Protocol Tab Select the Protocol tab in the User Preferences window to specify the default settings for a new protocol file in the Protocol Editor window e Protocol Editor Set the default settings that appear in the Protocol Editor Select a default sample volume to describe the volume of each sample in the wells and select a lid shutoff temperature e Protocol AutoWriter Selects default settings that appear in the Protocol AutoWriter Plate Tab Select the Plate tab in the User Preferences window Figure 77 to specify the following default settings for a new Plate file in the Plate Editor window e Plate Type Select the default plate type e Plate Size Select the default plate size e Units Select
16. 08064 Figure 68 Layout of the Gene Expression tab in the Data Analysis window TIP Right click on the chart to select right click menu options Select Sort from this menu to rearrange the order of the Target and Sample names in the chart Normalized Gene Expression To normalize data use the measured expression level of one or more reference genes targets as a normalization factor Reference genes are targets that are not regulated in the biological system being studied such as actin GAPDH or Histone H3 To set up normalized gene expression AAC t analysis follow these steps 1 2 Open a data file pcrd extension 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 Click the Gene Expression tab Choose a control in the Samples tab of the Experiment Settings window If a control is assigned the software normalizes the relative quantities for all genes to the control quantity which is set to 1 Select reference genes for this experiment in the Target tab of the Experiment Settings window Gene expression analysis requires one reference among the targets in your samples Select Normalized Expression AAC t if it is not already selected and then view the expression levels in the Gene Expression tab 81 Gene Expression Analysis 82 Relative Quantity Select Relative Quantity AC t from the pull down men
17. 54 30 61 Allele 1 Auto H04 28 39 37 87 Allele 1 Auto E04 29 02 38 77 Control 1 Auto FO4 29 07 38 69 Control 1 Auto G04 29 06 38 04 Control 1 Auto C t for Allele 2 VIC A03 34 14 28 40 Control Heterozyc Auto B03 33 58 28 16 Control Heterozyc Auto c03 33 68 28 15 Control Heterozyc Auto 405 33 86 24 90 Heterozygote Auto 40 BO5 32 62 23 91 Heterozygote Auto CO5 31 44 23 14 Het t Aut C t for Allele 1 FAM a a D03 32 26 27 29 Heterozygote Auto Allele 1 Control 4 A Control Heterozygote None DO5 34 35 25 17 Heterozygote Auto A Heterozygote EQ 50 00 50 00 None Auto Selected Fluorophores Allelel Y Allele2 x FAM Sa VIC v Call Selected Alleles v Display Mode Cct RFU Normalize Data Thresholds i Vertical 36 975 m Horizontal 28 724 Restore Default Thresholds Completed Scan Mode All Channels Plate Type BR White Analysis Mode Baseline Subtracted Curve Fit Figure 62 Layout of the Allelic Discrimination tab in the Data Analysis window CFX96 and CFX96 Deep Well Systems Instruction Manual Adjusting Data for Allelic Discrimination The software automatically assigns a genotype to wells with unknown samples based on the positions of the vertical and horizontal threshold bars and then lists genotype calls in the spreadsheet view To automatically call genotypes the software uses positive controls when available or estimates the thresh
18. All Wells C Time Course3 C Documents and Settings jlowery Desitop JOL GE 8 14 2007 11 36 01 AM All Wells Notes CFX Gene Study file Figure 71 Gene Study window 8 Gene Expression Analysis 88 Study Setup Tab Before importing data into a Gene Study do the following in the Data Analysis window Check that samples that contain the same content are named with the same name In a Gene Study the software assumes that wells with the same Target or Sample name contain the same samples Adjust the baseline and threshold C t in the Quantitation tab to optimize the data in 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 71 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 Remove experiments from this Gene Study Select one or more files in the list and click Remove Add notes about the Gene Study Type in the Notes box to add comments about the files and analysis in this Gene Study The Study Setup tab lists the data files in the Gene Study as described in Table 26 Table 26 Study Setup tab in Gene Study window Column Title Description File Name Name of the experiment data file pcrd extension File Folder Directory that sto
19. Status pane Displays the current progress of the protocol including the current step current GOTO repeat block temperature remaining hold time for the current step sample temperature lid and shuttle temperature e Run Status buttons Click one of the buttons to remotely operate the instrument or to interrupt the current protocol e Run Information pane Displays experiment details RUN STATUS TAB BUTTONS Click one of the buttons listed in Table 10 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 file associated with the run These actions are recorded in the Run Log Table 10 Run Status buttons and their functions Button Function Open the motorized lid on selected instruments WARNING Opening the lid during arun pauses the run during the current step and might alter the data Close the motorized lid on selected instruments 21 Running Experiments Table 10 Run Status buttons and their functions continued Button Function 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 Skip Step the software verifies that you want to skip the entire GOTO loop and proceed to the next step in the protocol R A lt Flash the LED on the selected instrument to i
20. 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 Standard Shows a standard curve with the threshold cycle plotted against the log of the starting quantity The legend shows the Reaction Efficiency E for each fluorophore in the wells with a standard sample type Well selector Selects the wells with the fluorescence data you want to show Spreadsheet Shows a spreadsheet of the data collected in the selected wells Fluorophore Selector To select the fluorophore data to display in the Quantitation tab charts and spreadsheets click the fluorophore selector below the Amplification chart Figure 53 Click the box next to the fluorophore name to show or hide the fluorophore data throughout the data analysis window gu FAM _ TET Figure 53 Fluorophore selector with FAM selected 65 Data Analysis Windows Trace Styles Window Open the Trace Styles window Figure 54 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 selection boxes 2 Click the Trace Styles button in the Data Analysis toolbar or select Settings gt Trace Styles in the Data Analysis menu bar Symbol Color a None AuWels Random Color anoe Eem Sho
21. 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 the Data Analysis window to display these data Choose one of these methods to open existing data files in the Data Analysis window e Drag a data file ocrd extension over the main software window and release it e Select File gt Open gt Data File in the main software window to select a file in the Windows browser e Click the Data Analysis button in the main software window toolbar to select a file in the Windows browser e Select File gt Recent Data Files to select from a list of the ten most recently opened data files The Data Analysis window displays up to nine tabs Figure 45 Each tab shows the analyzed data for a specific analysis method i ae E Quantitation Ve Quantitation Data Melt Curve zz Melt Curve Data alll Gene Expression eee End Point Allelic Discrimination Kh ac Py Run Information Figure 45 All the tabs that can display in the Data Analysis window The software only displays a tab in the Data Analysis window if the data are collected in the run and are available for that type of analysis Data Analysis Toolbar The toolbar in the Data Analysis window provides quick access to important data analysis functions Table 16 lists the functio
22. click the Show Details check box Table 25 also shows this information Table 25 Information in Gene Expression spreadsheet with Show Details selected Information Data Set Relative Quantity Relative Quantity SD Corrected Relative Quantity SD Unscaled Expression Unscaled Expression SD Corrected Unscaled Expression SD Expression Wells Description Fluorescence data from one fluorophore in the data file Calculated relative quantity of samples Standard deviation of the relative quantity calculation Calculated standard deviation of the corrected relative quantity Calculated unscaled expression Calculated standard deviation of the unscaled expression Corrected standard deviation of the unscaled expression Relative expression level Well number in the plate CFX96 and CFX96 Deep Well Systems Instruction Manual Experiment Settings Window Open the Experiment Settings window by clicking the Experiment Settings button in the Gene Expression tab In this window view or change the list of Targets and Samples select reference genes select control samples or set the Gene Expression Analysis sample group to be analyzed if Collection Names have been added to the wells Figure 69 Experiment Settings Targets Samples Name 4 Full Name Reference pea To 1 actin Actin m E 2 GAPDH GAPDH E 3 ILib IL1b E i 4 Tubulin Tubulin Add C Show Analysis Settings Sample Name Grouping Option Target vs Sample
23. current logged in software user is displayed at the top of the main software window Figure 73 Va Bio Rad CFX Manager Grant File View User Tools Windows aX 24 1 fa ew hire Figure 73 User name displayed CFX Manager software manages who logs in to the software through the Login dialog box Figure 74 When you start the software the Login dialog box opens automatically if there are two or more users listed in the User Administration window Bio Rad CFX Rad CFX Manager login R User Name Grant Password Figure 74 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 by clicking the Select User button in the toolbar or selecting User gt Select User in the menu bar 2 Select aname from the User Name pull down list The default is Admin administrator 3 Type a password in the Password box 4 Click OK to close the Login dialog box and open the software 91 Users and Preferences 5 To add a new user name and password contact your software administrator Change a Password Change a password by following these steps 1 Select User gt Change Password from the main software window menu to open the Change Password dialog box 2 Enter the old password in the Old Password box 3 Enter the new password in the New Password and the Confirm New Password boxes 4 Click OK to confirm the ch
24. e Remove the samples from the block and click the close lid button to close the lid of the CFX system e Press the power switch on the back panel of the C1000 thermal cycler to power down the system CFX96 and CFX96 Deep Well Systems Instruction Manual 2 CFX Manager Software Read this chapter for information about getting started with CFX Manager software e Main software window page 9 e Startup Wizard page 12 e Detected Instruments pane page 13 e Instrument Properties window page 14 Main Software Window Features available in the main software window are provided in Figure 9 Fa tte Rad CFX Manager Grant a eg Menu bar tie Yew User Toots widows Type word for Pew Toolbar at yO RD BIO RAD Detected Instruments pane Startup Wizard gt Oore new Loamert C Repos an Experemers J Open da Fie CJ Ooen a Gere SA Open U eee Prwtenena es Oweogtorn Set he protocel avd plate to begn an expeumers Status bar Figure 9 The main software window CFX Manager Software Menu Bar The menu bar of the main software window provides the items listed in Table 8 Table 8 Menu bar items in the main software window Menu Item Function File Create a new protocol plate experiment or Gene Study Open Open existing files including protocol prcl plate pltd data pcrd and Gene Study mgxd files LIMS plrn stand alone run files zpcr Recent Data Files View a list of the ten mo
25. ee Oe 62 Chapter 8 Data Analysis Windows 00000 cen e eee es 65 Q anitanon TaD eregtei aeee eee eed wah ee Gat ie Ee eclectic 65 Quantitation Data TaD aenea iba eee whee anes MAE Rea Rees 68 Mel Curie Wabi ovGrs 25avede4 65 E eb seees at Saad hea eee coarse eos 69 Melt Curve Data Tab 1 cc eee ees 70 EnA PON TAG stu tees eka eoe ceeds tae eee oed 4 65 08 Aaa bees 70 Allelic Discrimination 1abec lt ss4 3 wea hy ib we te Poke bee ee ya es A i 72 CNS Wd Ose aah te acase sane ape anne ie A 2 Meta Saute atin gece stents sn Ae ops sop park cee be ee os ee 74 RUPIMTORNMATION TaD had daaeie ae cee br eee Ma ee eae ee ee eS 74 Data File Repons eec canine vot et nea ee he Dae trae Peed oe E 75 Chapter 9 Gene Expression Analysis aaaaaan annann 79 Gene Expression wisc acca te vee eats ba abt e 6 ERa RERNE 79 Plate Setup for Gene Expression Analysis 00 0c eee eee eee eee 80 Gene Expression TaD r ims seanse a one ees Saeed aes a Pace baat does eke 80 Experiment Settings Window asaan cece eee eee eee 85 Gene Study ei bee vet OSU DD Na Oe ae a eee APR ad eel 86 Gene Study Data Spreadsheet 0 ccc eet ees 89 Gene study Report WindOW anana aana dae eid wewida ee eae eee Oded 90 Chapter 10 Users and Preferences ccc eee es 91 LOG In Or Select WS6ls c 22h eens eri eae a ha cares RFNA REREN 91 User Preferences Window 0 ccc cee eee eens 92 Configure Email Notification 0 0
26. for an instrument is the C1000 thermal cycler serial number which appears in many locations including the Detected Instruments pane Figure 12 To rename an instrument for ease of identification follow these instructions e In the Instrument Properties tab type a name in the Rename box at the top of the Properties tab and hit the Rename button to save the new name Instrument Properties CFX96SIM01 PoS Properties E Shipping Screw N Calibrated Dyes Rename your instrument Use alphabetic characters and numbers Click Rename button to apply Property Nickname Model Serial Numbers Base CFx9651M01 Block ALPA0123 Optics Shuttle SHUT0123 Optical Reaction Module HEAD0123 Firmware Yersions 1 0 113 0 1 35 1 0 242 1127 8051 Motorized Lid 50 Figure 12 Instrument Properties window CFX96 and CFX96 Deep Well Systems Instruction Manual Shipping Screw Tab The Shipping Screw tab includes instructions for installing or removing the red shipping screw To prevent damage to the optical reaction modules install the shipping screw any time you ship the CFX system NOTE If the shipping screw is detected by the software the Instrument Properties window automatically opens with the Shipping Screw tab in front Follow the instructions to remove the screw You can not perform a run with the shipping screw installed The information in this tab changes depending on whether the shipping screw is installed or removed For
27. g gt O Oo J CFX96 and CFX96 Deep Well Systems Instruction Manual Select the wells that will compose the well group in the plate view by clicking and dragging across the group of wells Selected wells turn blue in color Figure 29 Optional Change the name of the group by selecting the group name in the pull down menu and typing a new name Optional Create more well groups by repeating steps 1 and 2 Optional Delete well groups by selecting the group name in the pull down list and clicking the Delete button Click OK to finish and close the window or click Cancel to close the window without making changes Figure 29 Color of wells in the Well Group Manager window 41 Plates 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 30 by selecting Tools gt Show Spreadsheet View in the Plate Editor menu bar Plate Spreadsheet View fhos Fam ma g Unknown Unknoven Unknown Unknown Unknown Unknown Unknown Unknown Unknown Standard Standard Standard Standard Standard Standard Standard Standard 6 B 6 E 5 a D D D F F F F F F F G w Oman nwt wwf wn asa wn Ww A Column 4 Sample Type Impost Template Ke yy own f WO NH Tubulin Tubulin Tubulin Tubulin Tubulin Tubulin Tubulin Tubulin Tubulin Replicate TargetName Samp
28. maximum 80 non condensing e USB cable If the CFX system is going to be controlled by a computer via a USB cable the cable provided by Bio Rad is sufficiently shielded for use NOTE For a full list of the safety and compliance requirements for this instrument see Safety and Regulatory Compliance on page iii System Installation System Overview The CFX system includes two components e Optical reaction module This module includes an optical system to collect fluorescent data and a thermal cycler block NOTE The serial number of the CFX module is located on the back e C1000 thermal cycler base The C1000 base includes a user interface to control the system when running in stand alone mode and a power button and ports both on back panel to connect to a computer Indicator LED Open button Front panel Figure 1 Front view of the CFX system When open the CFX system includes the features shown in Figure 2 Close button Figure 2 Inside view of the CFX system WARNING Avoid touching the inner lid or block These surfaces can be hot CFX96 and CFX96 Deep Well Systems Instruction Manual e Inner lid with heater plate The heater lid maintains temperature on the top of the consumable to prevent sample evaporation Avoid touching or otherwise contaminating the heater plate Never poke anything through the holes the optics shuttle system could be damaged e Block Load samples in this block before th
29. of cleaning reagents WARNING Never clean the block with strong alkaline solutions Strong soap ammonia or concentrated bleach Never use corrosive or abrasive cleaning solutions These cleaning agents can damage the block and prevent precise thermal control WARNING Bleach ethanol or soap that is left in the blocks could corrode the block and destroy plastics during a run After cleaning always rinse the wells thoroughly with water to remove all traces of cleaning reagents WARNING Never heat the block after adding a cleaning solution Heating the block with cleaning solution will damage the block reaction module and thermal cycler base 105 Resources e Clean the inner lid Use a soft lint free cloth and water to remove debris and solutions from the inner lid surface Never use abrasive detergents or rough material that will scratch the surface Cleaning the inner lid improves precise sample heating and cooling Application Log Before a new run the instrument initiates a self diagnostic test to verify that it is running within the specifications The software records results of this test in the Run log and Application log file If you notice a problem in one or more experiments open the run and application logs to find out when the problem started CFX Manager software tracks information about the state of an instrument during a run in the Application Log Figure 83 Use these logs to track events that occur on instrume
30. selected instrument blocks Run Details Window When you click the Start Run button CFX Manager software prompts you to save the name of the data file and then opens the Run Details window Review the information in this window to monitor the progress of a run e Run Status tab Check the current status of the protocol open the lid pause a run add repeats skip steps or stop the run e Real Time Status tab View the real time PCR fluorescence data as they are collected e Time Status tab View a full screen countdown timer for the protocol 20 CFX96 and CFX96 Deep Well Systems Instruction Manual Figure 18 shows the features of the Run Details window a Run Details CFX Run CFX96SIMOO admin_2008 08 05 13 21 52 CFX96SIMOO pcrd Ral Run Status g Real time Status Time Status Run Status Run Information Protocol fast prel QuickPlate_96 wells_All Plate Channels pltd Sample Volume 25ul Scan Mode arte File sdmin_ 2008 08 05 ame 13 21 52_CFX96SIMO0 perd All Channels Step 3 of 4 65 0 C for 00 00 03 Sample 0 0 C Repeat 6 of 40 Remaining 00 35 30 Lid 105 C Running A Open Lid A Close Lid fig Add Repeats A Skip Step Flash Block Figure 18 Run Details window Run Status Tab The Run Status tab Figure 18 shows the current status of a run in progress in the Run Details window and provides buttons page 21 to control the lid and change the run in progress e Run
31. selected step 1 Click the Insert Melt Curve button CFX96 and CFX96 Deep Well Systems Instruction Manual 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 the Step Options button to enter the gradient range in the Step Options window page 29 NOTE You cannot insert a melt curve step inside a GOTO loop NOTE The melt curve step includes a 30 second hold at the beginning of the step that is not shown in the protocol Figure 22 shows a melt curve step added after step 6 8B Insert Step After Sample Volume ks Ju Run Time 01 34 00 95 0 C 3 00 1 95 0 C for 3 00 24 inset Step 5 2 0 Ct 010 3 550 C for 010 E Insert Gradient 4 720 C tor 030 Plate Read 5 GOTO 2 39 _ moretimes 6 95 0 C for 0 10 7 Met Curve Gait to 95 0 Lol E Af Figure 22 Protocol with inserted melt curve step Step Options To change a step option for the selected step 1 Select a step by clicking on the step in the graphic or text view 2 Click the Step Options button to open the Step Options window 3 Add or remove options by entering a number editing a number or clicking a check box TIP To hold a step forever an infinite hold enter zero 0 00 for the time Figure 23 shows the selected step with a gradient of 10 C Notice that some options are not available in a gradient step
32. the CFX optical reaction module into the reaction module bay of the C1000 chassis follow these instructions 1 Place the C1000 chassis in a suitable location with the locking bar down Remove any previously installed reaction modules System Installation 2 Lift the optical reaction module using the handle indents above the side air vents Figure 4 N opoe optical reaction module C1000 thermal cycler chassis Figure 4 Lifting the optical reaction module into the C1000 chassis 3 Position the module in the reaction 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 thermal cycler until it is flush with the sides of the module bay This action moves the module forward locking it into place Figure 5 i n j i pe M i i I i i ijji j I l Hl j A Whi W NW ii i iil Locking bar Figure 5 Locking the optical module into place 5 Check that the module is completely and evenly seated in the C1000 base There should be no extra space between the module and the base the space should be even CFX96 and CFX96 Deep Well Systems Instruction Manual 6 Plug the power cord into the back of the C1000 base Figure 3 on page 3 and into a
33. the Plate tab page 19 e To edit the current plate in the Plate tab click the Edit Selected button in the Plate tab page 19 e To open the plate associated with a data file in the Data Analysis window page 53 click View Edit Plate on the toolbar 33 Plates 34 Plate Editor GE_96 wells_ SYBR only pltd File Settings Tools B Zoom 100 Scan Mode All Channels fo Well Groups ita Plate Loading Guide HA Select Fluorophores Unk 2 unk 3 Unk 4 Unk 6 Unk7 Unke Unk3 Unk 10 Unk 11 unk az 2MPle Type k Ac yei pri G z GAPD peii pA E pa r Tubulin Tubulin iHr iHr 2Hr i Unk 3 Unk 4 teks nae 6 ae 7 ma 8 aie a Te 10 Unk 11 Unk 12 tin GAPDH GAPDH ILib Tubulin Tubulin Tubulin g OHr iHr 2Hr Unk 10 Unk 11 Unk 12 Tubulin Tubulin Tubulin OHr iHr 2Hr Pyar 8 Clear Replicate FS Clear Wells Plate Type BR Clear Figure 25 Plate Editor window Plate Size and Type The software applies these plate settings to all the wells during the experiment e Plate Size Select a Plate Size under Settings menu that represents the size of the reaction module block of your instrument Choosing the instrument type from the pull down menu option on the Startup Wizard will change the default plate size loaded in the Plate tab of the Experiment Settings window In the Plate Editor select the plate size from the Settings menu see Table 13 on page 35 Plate size cannot be change
34. the plate can be grouped into subsets for independent analysis using well groups When you create well groups in the Well Groups Manager window in the Plate Editor page 40 group names appear in the Data Analysis window within the Well Groups list on the toolbar By default the well group All Wells is selected when the Data Analysis Window is first opened with the data in all wells with content shown in the charts and spreadsheets Figure 48 shows Group 2 selected in the Well Groups menu Only the wells in that well group appear loaded in the well selector and data for these wells only are included in the data analysis calculations S a K A Gets View Edit Plate co WellGroup ED v O Quantitation Quantitation Data Melt Curve z5 Melt Curve Data Gene Expression 88 End Point S lt gt Amplification Standard Curve os 1D 15 20 25 30 35 Log Starting Quantity BOI SYBR Std5 802 SYBR Std 5 BO3 SYBR Std 5 BO4 SYBR Std 6 BOS SYBR Std 6 806 SYBR Std 6 807 SYBR Std 7 808 SYBR Std 7 A aa a e a R gt Figure 48 Data Analysis window with Group 2 selected Data Analysis Settings 58 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 fluorophore in one well These data are used to determine C t values for each well on a per fluorophore basis The software uses one of two mode
35. the protocol file into the Experiment Setup window and run it WARNING Always confirm the loaded protocol is correct in the Start Run tab before beginning the run Performing a run with the wrong protocol could alter the data or result in a failed run Opening the Protocol Editor To open the Protocol Editor follow one of these options To create a new protocol select File gt New gt Protocol or click the Create New button in the Protocol tab page 18 To open an existing protocol select File gt Open gt Protocol or click the Open Existing button in the Protocol tab page 18 To edit the current protocol in the Protocol tab click the Edit Selected button in the Protocol tab page 18 TIP To change the default settings in the Protocol Editor window enter the changes in the Protocol tab in the User Preferences window page 92 Protocol Editor Window The Protocol Editor window Figure 20 includes the following features Menu bar Select settings for the protocol Toolbar Select options for editing the protocol 25 Protocols e Protocol View the selected protocol in a graphic top and text bottom view Click the temperature or dwell time in the graphic or text view of any step to enter a new value e Protocol Editor buttons Edit the protocol by clicking one of the buttons to the left of the text view Protocol Editor CFX_2stepAmp prcl File Settings Tools B Insert Step After wv Sample Volume 25 ju
36. then click the Start Run button to begin the experiment Experiment Setup Options Add Protocol 223 Plate gt Start Run Run Information Protocol CFX_2stepAmp prcl Plate QuickPlate_96 wells_All Channels_pltd Notes Scan Mode All Channels Start Run on Selected Block s Block Name Type Status C48FSIMOO C4SFSIMO0 8 C48FSIMOO CFX96SIM01 CFX96SIMO1 SS6FSIMO2 S96FSIM02 C Select All Blocks A Flash Block Indicator Figure 17 The Start Run tab To add or remove run parameters from the spreadsheet in the Start Run on Selected Block s pane right click on the list and select an option in the menu to display Choose the value to change by clicking the text inside the cell to select it and then typing in the cell or by selecting a new parameter from the pull down menu Editable parameters include e Lid Temperature View the temperature of the lid Override the default lid temperature by selecting the text and typing a new temperature WARNING Changing the lid temperature can impact experiment results and may result in failed runs Buttons for Controlling the Instrument Click the following buttons in the Start Run tab to remotely operate the selected instruments e Start Run Start the experiment on the selected instrument blocks e Flash Block Indicator Flash the indicator LED on the selected instrument blocks e Open Lid Open motorized lid on selected instrument blocks e Close Lid Close motorized lid on
37. use the LIMS file to create a plate file that will be used in conjunction with the named protocol file to start a run and generate data The following steps should be performed by a LIMS specialist 1 Select the template file CFX96 LIMS Plate Import Template csv located in the SupportFiles folder C Program Files Bio Rad CFXIVD SupportFiles CFX 96 LIMS Plate Import Template csv Use a text editor to edit template file 2 Using the LIMS complete the template by filling in the required fields as listed in Table 29 3 Save the template with the file name extension plrn directly to your LIMS file folder location Note the pirn files and the protocol files referenced in the plrn files must exist in the same folder location 99 Resources 100 The plrn file is a format Known as a comma separated value CSV file It utilizes the comma as the separator between fields A comma should not be used within any field Changing the file extension from csv to plrn is required for CFX Manager software to recognize the file and start a LIMS run Floating point numbers are parsed as US English so the decimal mark used must be a period and not a comma Also for floating point numbers the thousands separator should not be used Table 29 Definition of LIMS csv file contents A Plate Header Do not edit Predefined A B D 2 Field Data Do not edit Predefined Instruction B Do not edit Predefined B Plate Size Do n
38. 50 Sample volume determines the Temperature Control mode page 30 Enter zero 0 to select Block mode Run Time View an estimated run time based on the Run Time 00 57 00 protocol steps and ramp rate D Open the software Help for more information about protocols L D Protocol Editor Controls The Protocol Editor window includes buttons for editing the protocol on the bottom left of the screen First select and highlight a step in the protocol by left clicking it with the mouse pointer Then click one of the Protocol Editor buttons at the bottom left side of the Protocol Editor window The location for inserting a new step Before or After the currently selected step is determined by the status of the Insert Step box located in the toolbar Insert Step Button To insert a temperature 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 Add or Remove a Plate Read To add a plate read to a step or to remove a plate read from a step 1 Select the step by clicking the step in either the graphical or text view 2 Click the Add Plate Read to Step button to add a plate read to the selected step If the step already contains a plate read the text on the button changes so now the same button reads Remove Plate Read Click to remove a plate read from the selected step 2 Proto
39. 977 6 340 589 6 528 302 and US application 2007246858 20080084004 20030180192 and 20060120927 Instruction Manual Information This manual contains information on how to safely set up and operate the CFX96 real time PCR detection system that carries the CE IVD mark catalog number 1855095 IVD as well as information on how to safely set up and operate the CFX96 Deep Well real time PCR detection system catalog number 1854095 IVD These two systems will be referred to as the CFX system in this instruction manual Additional information on CFX Manager software can be found in the CFX96 and CFX96 Deep Well Instruction Manual for life science research catalog numbers 1855095 1844095 Writing Conventions Used in this Manual The manual uses the writing conventions listed in Table 1 Table 1 Conventions used in this manual Convention Meaning TIP Provides helpful information and instructions including information explained in further detail elsewhere in this manual NOTE Provides important information including information explained in further detail elsewhere in this manual WARNING Explains very important information about something that might damage the researcher damage an instrument or cause data loss X gt Y Select X and then select Y from a toolbar menu or software window For information about safety labels used in this manual and on the CFX system see Safety and Regulatory Compliance on page iii Bio R
40. Clear Factory 5 30 2007 6 35 53 PM 12 Quasar 670 4 BR White Factory 5 30 2007 6 35 53 PM 13 Quasar 705 5 BR Clear Factory 5 30 2007 6 35 53 PM Figure 14 Calibrated Dyes tab in the Instrument Properties window 15 CFX Manager Software 16 CFX96 and CFX96 Deep Well Systems Instruction Manual 3 Running Experiments Read this chapter for information about running experiments using CFX Manager software e Experiment Setup window page 17 e Protocol tab page 18 e Plate tab page 19 e Start Run tab page 19 e Run Details window page 20 Experiment Setup Window The Experiment Setup window provides quick access to the files and settings needed to set up and run an experiment To open the Experiment Setup window follow one of these options e Click Create a New Experiment option in the Startup Wizard page 12 e Click the Experiment Setup button in the main software toolbar page 17 e Select File gt New gt Experiment in the main software menu bar page 10 The Experiment Setup window includes three tabs e 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 25 e 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 33 e Start Run Click the Start Run tab page 19 to check the run settings select one or more instrument blocks and begin the run NOTE
41. D window 3 Double click the CFX_Manager folder to open the folder and then double click setup exe to start the software installation wizard 4 Follow the instructions on the wizard to install the software and then click Finish 106 CFX96 and CFX96 Deep Well Systems Instruction Manual Power Failure Options In a power failure the instrument and computer will shut down After a short power failure the instrument will resume running a protocol but the Application log will note the power failure Depending on the computer settings and the length of time that the power is off the instrument and software attempt to continue running depending on the protocol step e f the protocol is in a step with no plate read then the protocol continues running as soon as the instrument gets power again e f the protocol is in a step with a plate read then the instrument waits for the software to restart and resume communication to collect the data In this situation the protocol only continues if the software is not shut down by the computer When the computer and software start up again 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 base 2 Place the module on the front of a desk so that the front of the m
42. DATA folder located in the SYSTEM folder Figure 41 FILE MANAGEMENT File Library Unit Wocons412 Se HA MAN E USE DRIVE H S PREVIOUS RUNS Ey SYSTEM JE RUN_LOGS E E RT_DATA li TEMPLATE Use the arrows on the keypad to navigate through the Ale tree Press ENTER to expand and collapse Folders SS i Leues E e ae j DE Tre a m Q aenani Figure 41 RT_DATA folder stores real time PCR runs lf a USB key has been placed in a USB key port on the C1000 thermal cycler the data zpcr will automatically be saved to the root directory of the USB key 49 Stand Alone Operation 50 lf a USB key is not in the thermal cycler at the end of the run follow these instructions i 2 3 5 Press the Files F2 button on the main screen to access the file folders Navigate to the RT_DATA folder and then press the right arrow key to open the folder Select the file using the up and down arrow keys Press Export File F1 to export the run data zpcr to the USB key Figure 42 FILE MANAGEMENT File Library File 20080730 075826 co Date a iz Z OF 02008 did Data Acquisition Mode 2 ep 20080730_111440_ce le TEMPLATE of USERS Use the arrows on the keypad to navigate through the file tree Press ENTER to expand and collapse Folders Figure 42 Exporting stand alone run data to a USB key Click Yes F1 to confirm the export You can choose to em
43. 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 Select the Gene Expression tab in the User Preferences window to specify the default settings for a new Gene Expression data file e Relative to Select a control or zero To graph the gene expression data originating at 1 relative to a control select Control When you assign a control sample in the 95 Users and Preferences 96 Experiment Setup window the software automatically defaults to calculate the data relative to that control Select Relative to zero to instruct the software to ignore the control which is the default selection when no control sample is assigned in the Experiment Settings window X Axis Graph the Target or the Sample on the x axis Y Axis Graph Linear Log 2 or Log 10 scale on the y axis Scaling Select a scaling option for the graph Leave the graph unscaled Alternatively choose a scaling option to scale to the Highest value or to the Lowest value Method Set the default analysis mode including normalized expression AACt or relative expression ACt Error Bar Select Std Dev for standard deviation or Std Error Mean for the standard error of the mean Std Devs Select the standard deviation multiplier to graph the error 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
44. Gene Expression graph e Standard Error of the Mean default SEMs e Standard Deviation Std Devs CHART ERROR BAR MULTIPLIER Select a multiplier for the error bars in the Gene Expression graph Select one of these integers 1 default 2 or 3 The type of multiplier changes when you select the Error Type e SEMs for Standard Error of the Mean e Std Devs for Standard Deviations TARGET STABILITY VALUE Open this window whenever more than 1 reference gene is used The software calculates two quality parameters for the reference genes e Coefficient of Variation CV of normalized reference gene relative quantities Lower CV values denotes higher stability e 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 Table 23 Table 23 Right click menu items Item Copy Save as Image Page Setup Print Show Point Values Set Scale to Default Chart Options Sort User Corrected Std Devs Use Solid Bar Colors x axis labels Function Copy the chart to a clipboard Save the graph in the chart view as an image file The default image type is PNG The other selections for image file types include GIF JPG TIF and BMP Select a page setup for printing Print the chart view Display the relative quantity of each point on the graph when you place the cursor over that point Set the c
45. Gene Study by adding data from one or more data files pcrd extension to the Gene Study the software groups them into a single file mgxd extension NOTE The gene expression data must include a common sample in every data file to create a Gene Study The software uses the common sample to normalize the data between experiments Select the sample names in the Experiment Settings window page 38 NOTE The maximum number of samples you can analyze in a Gene Study is limited by the size of the computer s RAM and virtual memory Gene Study Inter Run Calibration All data within the Gene Study are normalized by inter run calibrator to calculate the smallest average AC t value When the data files within the Gene Study include more than one inter run calibrator then the calibrator with the smallest average AC t value becomes the dominant inter run calibrator The dominant calibrator is used to adjust all C t values in the Gene Study CFX96 and CFX96 Deep Well Systems Instruction Manual To find the dominant inter run calibrator the software calculates the average of the AC t values for all inter run calibrators of 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 give
46. If the protocol currently selected in the Protocol tab does not include a step with a plate read for real time PCR analysis then the Plate tab is hidden To view the Plate tab add a Plate Read page 27 in at least one step in the protocol NOTE Start a new experiment from a previous run by selecting File gt Repeat an Experiment in the main software menu bar Then select the data file pcrd for the experiment you want to repeat 17 Running Experiments The Experiment Setup window opens with the Protocol tab in front Figure 15 To open another tab click that tab or click Prev and Next buttons at the bottom of the window Experiment Setup Options a Protocol EEF Plate gt Start Run CFX_2step4mp prel v Select Existing estep mp p Selected Protocol CFx_2step mp prel Edit Selected Preview Est Run Time 01 09 00 96 Wells All Channels Sample Volume 25ul Figure 15 Experiment Setup window including the Protocol Plate and Start Run tabs Protocol Tab 18 The Protocol tab shows a preview of the selected protocol file loaded in the Experiment Setup Figure 15 A protocol file contains the instructions for the instrument temperature steps and instrument options that control the ramp rate and lid temperature Select one of the following options to select an existing protocol create a new protocol or edit the currently selected protocol e Create New button Open the Protocol E
47. Maintenance 104 The CFX system includes a sensitive optical shuttle system and a sample block that must heat and cool very fast Contamination of these components can interfere with thermal cycling and data collection WARNING Never allow a reaction to run with an open or leaking sample lid The reagents could escape and coat the block inner lid and optical head in the shuttle system Excessive dirt can dim the signal and fluorescence contamination can create excessive background signal Only trained Bio Rad service engineers can clean the shuttle optical system Avoid contaminating the CFX system by following these suggestions e Always clean the outside of any container before placing it in the block e Never run a reaction with a seal that is open loose punctured or otherwise damaged doing so could contaminate the block inner lid and optical system e Never run a PCR or real time PCR reaction with volatile reagents that could explode and contaminate the block inner lid and optical system e Clean the block and inner lid periodically to prevent the buildup of dirt biohazardous material or fluorescent solutions page 105 e Never clean or otherwise touch the optical system behind the heater plate holes that are in the inner lid Figure 82 on page 105 e Clean the outer lid and C1000 thermal cycler base on a regular schedule for details see the C1000 thermal cycler instruction manual Cleaning the Optical Reaction Modul
48. QC rules to apply to data in Data Analysis Module The software validates the data against the enabled tests and the assigned values page 96 User Preferences Email Files a Protocol Ei Plate Fa Data nalysis al Gene Expression 7 Description Value UseRule Negative control with a Ct less than NTC with a Cft less than 38 NAT with a Cit less than 38 Positive control with a C t greater than 30 Unknown without a Cit Standard without a Cft Efficiency greater than 110 0 Efficiency less than 90 0 Std Curve R 2 less than 0 980 Replicate group Cit Std Dev greater than 0 2 Figure 79 QC tab in User Preferences Specify to add cut off values and to enable the following QC rules Negative control with a C t less than XX Input a C t cut off value NTC no template control with a C t less than XX Input a C t cut off value NRT no reverse transcriptase control with a C t less than XX Input a C t cut off value Positive control with a C t greater than XX Input a C t cut off value Unknown without a C t Standard without a C t Efficiency greater than XX Input a reaction efficiency cutoff value that is calculated for the standard curve CFX96 and CFX96 Deep Well Systems Instruction Manual e Efficiency less than XX Input a reaction efficiency cutoff value that is calculated for the standard curve e Std Curve R 2 less than XX Input a cutoff R 2 value for the standard curve e Replicate g
49. Use SSL Whether to use Secure Sockets Layer Some SMTP servers require this to be used others require that it not be used e 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 lt name gt YourCompany com If that is the case this checkbox must be unchecked and a valid from email address must be supplied in the box labeled From Address e Authentication Required Many SMTP servers require authentication If so this checkbox must be checked and a User Name and Password must be supplied e Test email To test the email settings enter one or more email addresses in Test Email Address text box Multiple email addresses can be separated by a comma Then click the Test Email button Options Outgoing Email SMTP ServerName mtp gmail com eg emto myCompany com Contact your IT Administrator to get the SMTP SEIVEr Name Port 25 4 Use SSL Use Default From Address Authentication Required UserName lt your account name gt gmail corn Password Test Email Address Test Attachment L Test Email Figure 76 Options to configure email NOTE Some SMTP servers do not allow attachments and others allow attachments only up to certain sizes If you will use CFX Manager software to email Data Files and or Reports you may want to test your server s ability to email
50. X NAT Load Target Name Fahd lt none gt HE none y ROS lt none gt wv Cys none v 7 Quasar 705 pore Load Load a Load Load Replicate a 1 z Replicate Series Replicate Group Size a i Starting Replicate 1 eh 123 Horizontal p a CO Vertical zA Cancel Apply Function After selecting wells the Sample Type must be loaded first Select a Sample Type from the pull down menu to load it in the selected wells including Unknown Standard NTC no template control Positive Control Negative Control and NRT no reverse transcriptase Click a Load box to add a fluorophore to the selected wells each fluorophore corresponds to a target name To add fluorophores to the Load list select them in the Select Fluorophores window To distinguish between multiple targets select a name in the Target Name pull down menu and press the Enter key to load the target name in the well To add a new target name to the pull down menu in the current plate only type a name in the pull down box and press the Enter key To delete a target name select it press the Delete key and press the Enter key For gene expression analysis or to distinguish between multiple samples select a Sample Name from the pull down menu to load that sample name in the selected wells To add a new sample name to the pull down menu in the current plate type a new name in the pull down bo
51. able 30 Table 30 Factory calibrated fluorophores channels and instruments Fluorophore Channel ae FAM SYBR Green 450 490 515 530 VIC HEX CAL Fluor Gold 540 Cal Fluor Orange 560 915 535 560 580 560 590 610 650 620 650 675 690 672 684 705 730 2 2 ROX Texas Red CAL 5 Fluor Red 610 TEX 615 CY5 Quasar 670 4 1 3 5 Quasar 705 Cy5 5 The CFX system also includes a channel dedicated for FRET chemistry this channel does not require calibration for specific dyes To open the Calibration Wizard to calibrate the CFX system 1 Select an instrument in the Detected Instruments pane 2 Select Tool gt Calibration Wizard to open the window and calibrate new dye and plate combinations Figure 81 102 CFX96 and CFX96 Deep Well Systems Instruction Manual Figure 81 shows an example of the Dye Calibration window Dye Calibration Base S N CFX96SIMO1 Optical Reaction Module S N HEADO1 23 Calibrated Fluorophores Fluorophore Channel Plate Type CalibratedBy 4 Date DeleteorRestore Enors Detail Calibrate New or Existing Fluorophores Fluorophore Piate Type Channel Plate column Fluor Color Add to Plate FAM BR Cle L zj 2 Settings Notes Calibration Status Ready to calibrate gt Calibrate 22 View Plate Figure 81 Dye Calibration window Calibrating the CFX System To calibrate the CFX system in the Dye Calibration window 1
52. ad Resources Table 2 lists Bio Rad resources and how to locate what you need Table 2 Bio Rad resources Resource How to Contact Local Bio Rad Laboratories Find local information and contacts on the Bio Rad website representatives 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 Type a search term in the Search box and select Literature to find links to technical notes manuals and other literature Technical specialists Bio Rad s Technical Support department is staffed with experienced scientists to provide customers with practical and expert solutions To find local technical support on the phone contact your nearest Bio Rad office For technical support in the United States and Canada call 1 800 424 6723 toll free phone and select the technical support option CFX96 and CFX96 Deep Well Systems Instruction Manual Safety and Regulatory Compliance For safe operation of the CFX system we strongly recommend that you follow the safety specifications listed in this section and throughout this manual The CFX96 system is approved for use as in vitro diagnostic IVD medical equipment Safety Warning Labels Warning labels posted on the instrument and in this manual warn you about sources of injury or harm Refer to Table 3 to review the meaning of each safety warn
53. ael 03 963 6050 Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 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 Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 026674 55 05 Taiwan 886 2 2578 7189 Thailand 1800 88 22 88 United Kingdom 020 8328 2000 10016715 RevC US EG 14 1953 1114 Sig 1213
54. ail your data to you directly from the C1000 thermal cycler after the run completes by configuring the email settings see the C1000 thermal cycler instruction manual for information on configuring the email settings To send an email with attached data zpcr at the end of a run follow these instructions 1 Select Options F4 in the Run information screen Figure 38 on page 48 Using the arrow keys select the Send email notification option 2 3 4 Click OK F1 to return to the Run information screen Use the arrow keys to navigate to the Email Address box and then use the alphanumeric keys to enter the email address CFX96 and CFX96 Deep Well Systems Instruction Manual 5 Click OK F1 to continue to run the assay Run C1O00RUN on ceiz 716 Ww Sample volume ji Lid Temperature Y ao me e Turn on hotlid User ADMIN Optional Email Address 9 XXXeXM BIO RAD COM Confirm settings and press RUM of OE to proceed KT ie BEEZ L U E irtual Figure 43 Confirming export to USB key Creating a Data File The stand alone run data zpcr data need to be converted into a data file pcrd by CFX Manager software to be analyzed 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 gt Open gt Stand alone Run from the main software window menu options to select the file n
55. ame 51 Stand Alone Operation 2 Inthe Run File Processor window click the Select Plate button to import the name of the plate file the software will use to create the data file Figure 44 Run File Processor 20080725_152353_CC003412_RIRUN zpcr x Run File Instrument Run Start Time Protocol E 420080725_152353_CC003412_RTRUN zper CC003412 7 25 2008 3 23 53 PM PCRUN prel lt None gt Select Plate Create Datafile Figure 44 Assigning a plate file NOTE CFX Manager software checks the scan mode and plate size for the plate file these must match the current run settings that were started during the experiment Load a Quick Plate file to quickly access data from all the wells 52 CFX96 and CFX96 Deep Well Systems Instruction Manual Data Analysis Overview Read this chapter for information about data analysis Data analysis precautions page 53 Data Analysis window page 54 Quantitation tab page 57 Data analysis settings page 58 Well selectors page 60 Charts page 62 Spreadsheets page 62 Data Analysis Precautions There are many ways in which the data generated from the system can be interpreted Occasionally the data can be interpreted incorrectly In order to prevent data misinterpretation please follow these guidelines when inferring a result from the data 1 O A O N Confirm that the sample data is correct If a barcode was entered check that the barcode of
56. ange 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 e Click the User Preferences button in the main software window toolbar e Select User gt User Preferences in the main software window menu bar e Click one of the tabs Figure 75 to view or change preferences User Preferences Email G Files a Protocol HB Plate Va Data Analysis alll Gene Expression Qc Email Notification Upon Run Completion Provide one email address per line Attach Data file or Run log C Attach Report file Configure Outgoing Email Outgoing email has not been configured Figure 75 User Preferences window with tabs Email Tab Select the Email tab Figure 75 to enter the email addresses where you want to receive confirmation of the completion of the run The software can send an attached data file or report file with the email when the check boxes next to these options are checked 92 CFX96 and CFX96 Deep Well Systems Instruction Manual Configure Email Notification Click the Configure Outgoing Email button to open the Options window Figure 76 to configure the SMTP server and send a test email from the computer Input the following e SMTP Server Name The name of the SMTP server as provided by your ISP e Port The port number of your SMTP server as provided by your ISP this is usually 25 e
57. ata in relative fluorescence units at the selected cycle e Select Normalize Data to normalize the RFU data shown in the chart and spreadsheet Normalization changes the data on the chart to a range from 0 to 1 on both axes To normalize the data the plate must contain wells with no template control NTC sample types for both Allele 1 and Allele 2 For this plot the RFU data are normalized to the NTC values as a linear combination of Allele 1 and Allele 2 specific RFUs This plot is an effective way to present RFU data The calculations for normalized RFUs follow the formulas presented in Livak et al 1995 A NoiMdlized Ay e M A4 A X NTC h4 ao Where e A represents RFU for Allele 1 e A represents RFU for Allele 2 e X represents the mean RFU NTCa4 ao represents the sum of RFUs for the NTC sample of Allele 1 and Allele 2 13 Data Analysis Windows QC Tab Open the QC tab to quickly assess the quality of the experimental data based on the rules defined in the QC tab in the User Preferences window see QC Tab on page 96 The software displays the currently applied QC rules and the settings that define each rule Figure 63 The rule description also displays wells that fail a selected rule NOTE You can turn on or turn off rules by clicking the check box next to the rule in the Use Rule column Amplification miojnjoj r Run information Tab The Run Information tab Figure 64 shows the prot
58. ate create a new plate or edit the currently selected plate e Create New button Open the Plate Editor to create a new plate Select Existing button Open a browser window to select and load an existing plate file pltd extension into the Plate tab Express Load pull down menu Quickly select a plate to load it into the Plate tab TIP To add or delete plates in the Express Load menu add or delete files pltd extension in the Express Load folder To locate this folder select Tools gt User Data Folder in the menu bar of the main software window Edit Selected button Open the currently selected plate in the Plate Editor a Protocol i3 Plate gt Stat Run e q pit Select Existing QuickPiste pltd Selected Fiste v QuickPlate pltd Edk Selected Preview Fluorophores FAM HEX ROX Cy5 Quasar 705 Plate Type BR White Scan Mode All Channels Figure 16 Plate tab window Start Run Tab The Start Run tab Figure 17 includes a section for checking information about the run that is going to be started including the selected protocol and plate files and a section for selecting the instrument block e 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 19 Running Experiments e Start Run on Selected Block s pane Select one or more blocks edit run parameters if necessary and
59. ce with laboratory local regional and national regulations e Clinical samples e Reagents e Used reaction vessels or other consumables that may be contaminated Chemical Hazards The CFX system contains no potentially hazardous chemical materials Explosive or Flammability Hazards The CFX system poses no uncommon hazard related to flammability or explosion when used in a proper manner as specified by Bio Rad Laboratories Electrical Hazards The CFX system poses no uncommon electrical hazard to operators if installed and operated properly without physical modification and connected to a power source of proper specification Transport Before moving or shipping the C1000 thermal cycler or optical reaction module decontamination procedures must be performed Always move or ship the C1000 thermal cycler chassis and optical reaction module in separate containers with the supplied packaging materials that will protect the instrument from damage If appropriate containers cannot be found contact your local Bio Rad office Storage The CFX system can be stored under the following conditions e Temperature range 20 to 60 C e Relative humidity maximum 80 Disposal The CFX system contains electrical or electrical materials it should be disposed of as unsorted waste and must be collected separately according to the European Union Directive 2002 96 CE on waste and electronic equipment WEEE Directive Before disposal contact your l
60. cols 28 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 Edit the gradient temperature range by clicking the default temperature in the graphic or text view and entering a new temperature 3 Edit the hold time by clicking the default time in the graphic or text view and entering a new time Figure 21 shows the inserted gradient step The temperatures of each row in the gradient are charted on the right side of the window BJ Insert Step After v Sample Volume es ul Run Time 01 28 00 05 E 3 00 65 0 1 95 0 C for 3 00 Gradient AS Inset Step 2950 Cfo ene foi Add Plate Read to Step Delete Step Figure 21 Protocol with inserted gradient step p 3 radient 33 U 4 Del Insert Gradient 4 55 0 C for 0 30 X Plate Read B ee 5 GOTO 2 39 moe times Ke TEND SOCOCOCCCCCCSC S r E F G H Insert GOTO Button To insert a GOTO step before or after the selected step 1 Click the Insert GOTO button 2 Edit the GOTO step number or number of GOTO repeats by clicking the default number in the graphic or text view and entering a new value Figure 21 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
61. control in the Experiment Settings window Expression Normalized Gene Expression AAC t or Relative Quantity AC t depending on the selected mode Expression SEM or SD Standard Error of the Mean or Standard Deviation depending on the selected option 89 Gene Expression Analysis Table 27 Information in the spreadsheet on the Study Analysis tab continued Information Description Corrected Expression SEM _ Corrected value calculation for Standard Error of the Mean or SD 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 Show Details Data Click the Show Details check box to show additional information The spreadsheet adds the information in the columns listed in Table 28 Table 28 Information added to the spreadsheet when Show Details selected Information Description Data Set Fluorescence data from one fluorophore in one data file Relative Quantity Calculated relative quantity of samples Relative Quantity SD Standard deviation of the relative quantity calculation Corrected Relative Quantity Calculated standard deviation of the corrected relative quantity SD Unscaled Expression Calculated unscaled expression Unscaled Expression SD Calculated standard deviation of the unscaled expression Corrected Unscaled Cor
62. d behind the heater plate holes in the inner lid Figure 82 Never touch anything behind these holes Block 96 well Figure 82 Heating plate holes in the inner lid Clean the outer surface Use a damp cloth or tissue to clean spills from the outside case If needed use a mild soap solution and rinse the surface with a damp cloth Clean the cover to prevent corrosion Clean the cooling fins Remove dust with a soft brush or damp cloth Remove any heavy dust that is deep in the vents with a vacuum cleaner Use water and a soft lint free cloth to remove debris that is stuck to the fins Avoid scratching the surface If needed use a mild soap solution and rinse well to remove residue completely Cleaning the fins improves precise 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 block Clean spills immediately to prevent them from drying Use disposable plastic pipets with water recommended 95 ethanol or a 1 100 dilution of bleach in water Also use a soft lint free cloth or paper towel and water to clean the block Always rinse the wells with water several times to remove all traces
63. d during or after the experiment e Plate Type Select Clear Wells or White Wells from the Plate Type under the Settings menu Make sure the fluorophore being used in the experiment is calibrated for the selected plate type NOTE CFX 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 gt Calibration Wizard see Calibration Wizard on page 99 Scan Mode The CFX system excites and detects fluorophores in six channels and uses multiple data acquisition scan modes to collect fluorescence data from during a run Select one of these scan modes in the Plate Editor window toolbar CFX96 and CFX96 Deep Well Systems Instruction Manual e All Channels Includes channels 1 through 5 on the CFX system e SYBR FAM only Includes only channel 1 and provides a fast scan e FRET Includes only the FRET channel and provides a fast scan Plate Editor Toolbar The toolbar in the Plate Editor provides quick access to important plate loading functions Table 13 lists the functions available in the Plate Editor toolbar Table 13 Toolbar items in the Plate Editor Toolbar Item Function Save Save the current plate file Print the selected window Increase or decrease magnification in plate view Select a scan mode to instruct the instrument what channels to collect fluor
64. d open it in the Data Analysis window page 53 Open a Gene Study Open a browser window to locate a Gene Study file mgxd extension and open it in the Gene Study window page 87 Create a New Gene Open the Gene Study window page 87 to add files Study and create a new study Print Print the current software window Startup Wizard Open the Startup Wizard that links you to common software functions page 12 11 CFX Manager Software Startup Wizard 12 Table 9 Toolbar buttons in the main software window Button Button Name Function Experiment Setup Open the Experiment Setup window to run an experiment page 17 Protocol AutoWriter Open the Protocol AutoWriter window to create a new protocol page 30 Select User Open the Select User window to change software users see Log In or Select User on page 91 User Preferences Open the User Preferences window page 92 Open the software Help window for more information about running PCR and real time PCR The Startup Wizard automatically appears when CFX Manager software is first opened Figure 9 If it is not shown click the Startup Wizard button on the main software window toolbar Options in the Startup Wizard include the following e Create a new Experiment page 17 Set up the protocol and plate to begin a new experiment e Repeat an Experiment Set up an experiment with the protocol and plate from a completed run If needed you can edi
65. dentify the y Ha Block selected blocks ndicator Pause the protocol j Pause NOTE This action is recorded in the Run Log Resume a protocol that was paused ae Resume Stop the run before the protocol ends p stop WARNING Stopping a run prematurely may alter your data and may result in a failed experiment Real Time Status Tab The Real time Status tab Figure 19 shows real time PCR data collected at each cycle during the protocol after the first two plate reads This tab also shows the well selector and text describing the protocol status at the bottom of the window Run Details CFX Run CFX96SIMOO admin_2008 08 05 13 21 52 _CFX96SIMOO pcrd Run Status GQ Real time Status Time Status Amplification FAM HEX TexasRed C5 Quasar 705 Step Number 3fAmp 2 3 4 5 8 9 10 11 12 1 a ac o our p unk p unk p ork p ork p ork p ork p ork uk ok ok unk _ un p unk p or p ork p ork p ork p ork p ork ork ok ok ork _ D Unk Unk Unk Il Unk II Unk II Unk II Unk II Unk II Unk II Unk II Unk II Unk l E F G H unk J Unk JP unk Il Unk ll Unk l Unk Unk Unk il Unk Unk Unk Unk l unk uk I uk I un u If uk unk I uk If unk J u I unk J unk unk unk f o p unk If rk If rk If uk If uk If uk uk uk u Unk ff unk funk Punk Junk unk Punk Punk Jf unk Jf unk nk nk Step 3 of 4 65 0 C for 00 00 01 Sample 0 0 C Repeat 8 of 40 Remai
66. ditor to create a new protocol e 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 Express Load folder To locate this folder select Tools gt User Data Folder in the menu bar of the main software window e Edit Selected button Open the currently selected protocol in the Protocol Editor End Point Only Runs To run a protocol that contains only an end point data acquisition step select Options gt End Point Only Run from Options in the menu bar of the Experiment Setup window The default end point protocol which includes two cycles of 60 0 C for 30 seconds is loaded into the Protocol tab To change the step temperature or sample volume for the end point only run click the Start Run tab and edit the Step Temperature or Sample Volume CFX96 and CFX96 Deep Well Systems Instruction Manual Plate Tab The Plate tab shows a preview of the selected plate file loaded in the Experiment Setup Figure 16 In a real time PCR experiment the plate file contains a description 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 pl
67. 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 cable into the USB 2 0A port located on the back of the chassis Figure 3 on page 3 and then connecting the cable into the USB 2 0 B port located on the computer If it is not already turned on turn on the system using the power switch on the back of the C1000 chassis Follow the instructions in the Found New Hardware Wizard that launches after the instrument is first detected by the computer On the first screen select Yes this time only to instruct the Windows operating system to connect to Windows Update to search for software Click Next Instruct the wizard to Install the software automatically Click Next to continue installing the drivers Click Finish at the software installation completion screen when the drivers are installed CFX96 and CFX96 Deep Well Systems Instruction Manual Software Files CFX Manager software stores information about experiments in specific files Table 7 Table 7 Open these file types with CFX Manager software File Type Extension How to View and Edit File Protocol Select in Experiment Setup and edit in Protocol Editor Plate Select in Experiment Setup and edit in Plate Editor Data View and analyze in Data Analysis window LIMS Contains plate setup and protocol info
68. e The block of the optical reaction and the C1000 thermal cycler base should be cleaned ona regular schedule to remove any debris or dirt that might interfere with proper function Clean as soon as you discover debris and spilled liquids with a soft lint free cloth that is dampened with water Cleaning the instrument allows precise instrument function For more detailed information about cleaning the C1000 base see the C1000 thermal cycler instruction manual WARNING Never use cleaning solutions that are corrosive to aluminum Avoid scratching the surface of the C1000 reaction module bay Scratches and damage to this surface interfere with precise thermal control WARNING Never pour water or other solutions in the C1000 reaction module bay Wet components can cause electrical shock when the thermal cycler is plugged in Clean the CFX optical reaction module as soon as you discover debris dirt or contamination in the block or on the inner lid Any dirt can interfere with the ability of the block to change temperature quickly and collect accurate fluorescent data To clean the reaction module follow these guidelines Follow these suggestions for cleaning WARNING To prevent electrical shock always remove the reaction module from the thermal cycler base or unplug the base before cleaning the instrument CFX96 and CFX96 Deep Well Systems Instruction Manual WARNING Never touch or allow solutions to touch the optical system that is locate
69. e List a view of the data and contents of each well in the plate including the content sample name peak 1 and peak 2 e RFU List the RFU quantities at each temperature for each well e d RFU dT List the negative rate of change in RFU as the temperature T changes for each well This is a first regression plot for each well in the plate End Point Tab Open the End Point tab to analyze final relative fluorescence units RFUs for the sample wells The software compares the RFU levels for wells with unknown samples to the RFU levels for wells with negative controls and scores the unknown as a Positive or Negative Positive samples have an RFU value that is greater than the average RFU value of the negative controls plus the Cut Off Value To analyze the end point data the plate must contain negative controls or the software cannot make the call Run one of these two types of protocols e 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 70 CFX96 and CFX96 Deep Well Systems Instruction Manual Run an End Point Only protocol Load the End Point Only protocol in the Plate tab of the Experiment Setup window select or create a plate and run the experiment The End Point tab shows the average RFU values to determine whether or not the target was amplif
70. e run e Close button Press this button on the inside of the lid to close the motorized lid WARNING Prevent contamination of the instrument by spills and never run a reaction with an open or leaking sample lid For information about general cleaning and maintenance of the instrument see Instrument Maintenance page 101 The back panel of the C1000 chassis includes these features Figure 3 e Power switch Press the power switch to turn on the power to the system e Power input Plug in the power cord here e Ethernet port Connect an ethernet cable to email run logs and stand alone data files e USB connections Use these ports to connect the CFX system to a computer or to connect an S1000 thermal cycler Power Power Ethernet switch input port USB connections GS x y ae f i yae EE l f ae z Bl OCBC J IVEUSSSeeese 1 Figure 3 Back panel of C1000 thermal cycler A WARNING Do not touch the back of the CFX system when the instrument is on Setting Up the System The CFX system should be installed on a clean dry level surface with sufficient cool airflow to run properly The CFX system can be run in two modes stand alone or software controlled If you are running the system under software controlled mode make sure there is sufficient space for a computer during setup Position the C1000 thermal cycler base so that the power switch which is located on the back panel is easily accessible To insert
71. e 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 8 Click the OK F1 button to start the run Running a Previously Saved Protocol e To change an existing protocol press the EDIT key to open the file library and select a protocol to edit 48 CFX96 and CFX96 Deep Well Systems Instruction Manual e Torun an existing protocol press the RUN command key and select a previously saved protocol from the file library Monitoring a run When a run begins the run status window appears Review the information in this window to monitor the progress of a run e Status Press the STATUS command key to check the current status of the protocol pause the run cancel a run skip a step or access the main menu Figure 40 e Time Status Press the VIEW command key to see a full screen count down timer for the protocol Press the VIEW key again to switch back to the Status screen Protocol RUNA Cycler CCOOS417 User SamplelD 10 Step 1 of 3 Lid Preheating Sample 259 C Repeat 1 of 11 Remaining 00 13 15 Lid 51 72 Yoluroe 1Opl Shuttle 37 770 _ lt lt o S _ m lt Figure 40 Monitoring run status Exporting Data for Analysis When the run is finished the fluorescence data need to be transferred to a computer running CFX Manager software for analysis The stand alone data file is automatically saved to the RT_
72. el Protocol pPHanurieie AutoWriter key LCD e C1000 Thermal Cycler eee A Navigation aa ie LN keys eys 000 USB port below Function keys Figure 31 The C1000 thermal cycler control panel 43 Stand Alone Operation 44 The control panel contains five sets of keys with the functions listed in table Table 15 Table 15 Functions 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 key multiple times to switch to each associated letter 0 INCUBATE Insert a zero infinity or start instant incubation decimal point Enter a decimal point minus sign Enter a minus sign Protocol AutoWriter Protocol AutoWriter key Launch the Protocol AutoWriter NAVIGATION KEYS RIGHT arrow Move cursor to the right LEFT arrow Move cursor to the left UP arrow Move cursor up DOWN arrow Move cursor down ENTER Confirm a setting BACK Cancel a function Delete a letter number or word Main Menu When the CFX system starts it runs a self test to verify proper functions and then displays the main menu The main menu provides access to all syste
73. ents serial numbers Zip Data and Log Choose and condense selected files in a zipped file for Files storage or to email Configure software email 10 CFX96 and CFX96 Deep Well Systems Instruction Manual Table 8 Menu bar items in the main software window Menu Item Windows Help Command Function Cascade Arrange software windows on top of each other Tile Vertical Arrange software windows from top to bottom Tile Horizontal Arrange software windows from right to left Close All Close all open software windows Contents Open the software Help for more information about running PCR and real time PCR Index View the index in the software Help Search the software Help Gene Expression Open a website to find information about running PCR Gateway Website and real time PCR experiments PCR Reagents View a website that lists Bio Rad consumables for PCR Website and real time PCR reagents PCR Plastic View a website that lists Bio Rad consumables for PCR Consumables and real time PCR experiments Website Software Updates Check for software updates from Bio Rad Open a window to see the software version Toolbar Buttons Click a button in the toolbar of the main software window Table 9 for quick access to common software commands Table 9 Toolbar buttons in the main software window Button w Button Name Function Open a Data File Open a browser window to locate a data file pcrd extension an
74. escence data from during a run Select All Channels default SYBR FAM only or FRET GA Plate Loading Suide Select Fluorophores Window The Select Fluorophores window lists fluorophores that can be selected to load into the Plate Editor well loading controls To open the Select Fluorophores window click the Select Fluorophores button on the right side of the Plate Editor 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 99 After calibration the new fluorophore is added to the Select Fluorophore window 35 Plates Click the Selected check box next to the fluorophore name to add or remove the fluorophores to the list on the right side of the Plate Editor window In this example SYBR is selected from the list of available fluorophores Figure 26 Select Fluorophores Channel FAM SYBR Cal FL Gold Cal FL Red SBG1 HEX TET Cal Gold 540 VIC TexasRed ROX Texas Red Cal Red 610 cys Quasar 670 Quasar 705 Selected a et an a a a Figure 26 Select Fluorophores window e Click the Color box next to the fluorophore name and select a new color to represent each fluorophore in the Plate Editor window and Data Analysis charts NOTE
75. etermine threshold cycles construct standard curves and determine the concentration of unknown samples To generate a baseline subtracted trace the software fits the best straight line through the recorded fluorescence of each well during the baseline cycles and then subtracts the best fit data from the background subtracted data at each cycle e Baseline Subtracted Curve Fit The software displays the data 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 e To hide one well highlight and click the individual well To show that well highlight and click the well again e To hide multiple wells click and drag across the wells you want to select To show those wells click and drag across the wells again e Click the top left corner of the plate to hide all the wells Click the top left corner again to show all wells e Click the start of a column or row to hide those wells Click the column or row again to show the wells Only wells loaded with content entered in the Plate Editor can be selected in the well selector and their color shows if they are selected As shown in Figure 50 the well selector shows these three types of wells e Selected loaded wells blue
76. ets and samples in the Experiment Settings window e Show Details check box Click Show Details to add more columns of data to the chart Highlighting a sample in the Gene Expression chart highlights the corresponding cell in the spreadsheet below the chart Figure 72 23 Study Setup ail Study Analysis Gene Expression Mode A Normalized expression AAI w Graph Data Eirce Type Standard Deviation v Chast Error Bar Multipher 1 S StdDevs 5Hr 6Hr 7Hr SHr C Show Detais v ted S oe a x a Bz ao SS E o z Coerected A Taget amp Sampe gt Ctl Expesion ene Expression Meant Chisd gt 0 Tubulin 4H 210 46245 28 67067 28 67067 11 65 0 15948 Tubulin 5H 832 66707 145 87741 145 97741 9 62 0 17616 Tubulin 6Hr 4672 54256 523 10429 523 10429 7 52 0 12490 Tahi n Th LAITA ATNA NAN INTA NA INTA Ran NIRA Figure 72 Study Analysis tab in Gene Study window Gene Study Data Spreadsheet The data spreadsheet in the Gene Study window lists information about each target and sample in the Gene Study Figure 72 Table 27 describes the information shown in the Gene Study spreadsheet Table 27 Information in the spreadsheet on the Study Analysis tab Information Description Target Target Name amplified 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
77. example to install the shipping screw click the Install Shipping Screw button and follow the instructions in the tab Figure 13 Instrument Properties CFX96SIM01 A Properties ka Shipping Screw N Calibrated Dyes Shipping Screw is removed Instructions 1 Click Install Shipping Screw Install Shipping Screw X 2 Open the Optical Module iid 3 Insert the shipping screw into the hole in the inner heated lid that conesponds to well A1 4 Click OK to confirm the screw status Figure 13 Instructions for installing the shipping screw Calibrated Dyes Tab Open the Calibrated Dyes tab to view the list of calibrated fluorophores and plates for the selected instrument Figure 14 Click an Info button to see detailed information about a calibration Instrument Properties CFX96SIMO0 ey Properties E Shipping Screw N Calibrated Dyes Fluorophore lt gt Channel PlateType CalibratedBy Date Eros Detail 1 CalFLGold 1 BR Clear Factory 5 30 2007 6 35 53 PM 2 CalFL Gold 1 BR White Factory 5 30 2007 6 35 53 PM 3 CalFL Red 1 BR Clear Factory 5 30 2007 6 35 53 PM 4 CalFL Red 1 BR White Factory 5 30 2007 6 35 53 PM 5 Cy5 4 BR Clear Factory 5 30 2007 6 35 53 PM 6 jCy5 4 BR White Factory 5 30 2007 6 35 53 PM 7 FAM 1 BR Clear Factory 5 30 2007 6 35 53 PM g FAM 1 BR White Factory 5 30 2007 6 35 53 PM g HEX 2 BR Clear Factory 5 30 2007 6 35 53 PM 10 HEX 2 BR White Factory 5 30 2007 6 35 53 PM 11 Quasar 670 4 BR
78. fectious material e Upon completion of the operation involving biohazardous material decontaminate the work area with an appropriate disinfectant for example a 1 10 dilution of household bleach SPECIFIC PRECAUTIONS e All patient samples are a potential biohazard and should be handled accordingly using universal precautions e No biohazardous substances are exhausted during normal operations of this instrument SURFACE DECONTAMINATION WARNING To prevent electrical shock always turn off and unplug the instrument prior to performing decontamination procedures The following areas can be cleaned with any hospital grade bactericide virucide or fungicide disinfectant e Outer lid and chassis e Inner reaction block surface and reaction block wells e Control panel and display To prepare and apply the disinfectant refer to the instructions provided by the product manufacturer Always rinse the reaction block and reaction block wells several time with water after applying a disinfectant Thoroughly dry the reaction block and reaction block wells after rinsing with water WARNING Do not use abrasive or corrosive detergents or strong alkaline solutions These agents can scratch surfaces and damage the reaction block resulting in loss of precise thermal control DISPOSAL OF BIOHAZARDOUS MATERIAL The CFX system contains no potentially hazardous chemical materials Dispose of the following potentially contaminated materials in accordan
79. fine the type of information that appears in any report if you save the report as a template Select Template gt Save or Save As to save the layout of the current report as a template Create a Data Analysis Report To create a report in the Data Analysis window follow these steps 1 Make final adjustments to the well contents selected wells charts and spreadsheets in the Data Analysis window before creating the report 2 Click the Report button in the Data Analysis toolbar to open the Report window 3 Change the options you want to include in the report The report opens with default options selected Click the check boxes in the report 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 19 Data Analysis Windows 76 4 Click the Update Report button to update the Report Preview with any changes 5 Print or save the report Click the Print button in the toolbar to print the current report Select File gt Save to save the report as a PDF Adobe Acrobat Reader file MHT Microsoft document or MHTML Microsoft document formatted file and select a location to store the file Select File gt Save As to save t
80. g of materials IEC 61010 2 081 2001 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 e EN 61326 1 2006 Class A Electrical equipment for measurement control and laboratory use EMC requirements Part 1 General requirements e EN 61010 2 101 Safety requirements for electrical equipment for measurement control and laboratory use Particular requirements for in vitro diagnostic IVD medical equipment EN 61326 2 6 Class A Electrical equipment for measurement control and laboratory use EMC requirements Part 2 6 Particular requirements In vitro diagnostic IVD medical equipment CFX96 and CFX96 Deep Well Systems Instruction Manual This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense Hazards The CFX system is designed to operate safely when used in the manner prescribed by the manufacturer If the system or any of its associated components are used in a manner not specified by the manufacturer the inherent protec
81. hart view back to the default settings after magnifying it Open the Chart Options window to adjust the graph Sort the order that samples or targets appear on the chart x axis Calculate the error bars using the corrected standard deviation formula Display solid bars in the graph Choose to display x axis labels horizontal or angled 83 Gene Expression Analysis 84 Gene Expression Spreadsheet Table 24 describes the information shown in the Gene Expression spreadsheet Table 24 Description of information in the spreadsheet on the Gene Expression tab Information Target Sample Ctrl Expression Expression SEM or SD Corrected Expression SEM or SD Mean C t C t SEM or SD Show Details Option Description Target Name amplified gene selected in the Experiment Settings window Sample Name selected in the Experiment Settings window Control sample when the Sample Name is selected as a control in the Experiment Settings window Normalized Gene Expression AAC t or Relative quantity AC t depending on the selected mode Standard Error of the Mean or Standard Deviation depending on the selected option Corrected value calculation for Standard Error of the Mean SEM or Standard Deviation SD of the relative expression depending on the selected option Mean of the threshold cycle Standard Error of the Mean or Standard Deviation of the threshold cycle depending on the selected option When you
82. he report with a new name or ina new location 6 Optional Create a report template with the information you want To save the current report settings in a template select Template gt Save or Save As Then load the report template the next time you want to make a new report Data Analysis Report Categories A report can include any of the options in each category described in Table 22 depending on the type of data in the Data Analysis window Table 22 Data analysis report categories in the options list Header Title subtitle and logo for the report Report Information Experiment date user name data file name data file path and selected well group Notes about the data report Run Information Includes the experiment date user data file name data file path and the selected well group Experiment Setup Protocol Text view of the protocol steps and options Plate Display Show a plate view of the information in each well of the plate Quantitation Analysis Settings Includes the step 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 Spreadsheet listing the data in each well Analysis Settings Includes the analysis mode chart data scaling option and chart error Copy of the gene expression chart Target Names Char
83. ht click on the spreadsheet to select one of these options from the right click menu 42 Copy Copy the entire spreadsheet Copy as Image Copy the spreadsheet as an image file Print Print the spreadsheet Print Selection Print only the selected cells Export to Excel Export the file as an Excel formatted file Export to Text Export the file as a text file Find Find text in the spreadsheet Sort Sort the spreadsheet by selecting up to three columns of data in the Sort window CFX96 and CFX96 Deep Well Systems Instruction Manual 6 Stand Alone Operation Read this chapter for information about running the CFX system in stand alone mode e C1000 thermal cycler page 43 e Creating a new experiment page 45 e Exporting data for analysis page 49 e Creating a data file page 51 C1000 Thermal Cycler The CFX 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 the C1000 thermal cycler Instruction Manual for information on how to configure the email settings The data requires CFX Manager software for analysis The control panel on the C1000 thermal cycler provides access to all the functions needed to run the instrument Figure 31 shows the components of the control pan
84. ical set name Optional Name Q Replicate Enter a positive integer for each Optional set of replicates The value cannot be zero R W CH1 Quantity Enter quantity values for any Required CH2 Quantity standards for all CH3 Quantity Enter concentration in decimal standards CH4 Quantity form CH5 Quantity FRET Quantity Y AD Chi Well Color Enter any user defined trace Not Ch2 Well Color Ch3 Well Color Ch4 Well Color style color enabled in a 32 bit integer ARGB decimal format Ch5 Well Color FRET Well Color Initiating a LIMS Run To initiate a LIMS run 1 Open a LIMS file using one of the following methods 101 Resources e Drag and drop the plrn file onto the CFX Manager software window or desktop icon e Double click on the desired plrn file e Select File gt Open gt LIMS file from the main software window menu bar In the Experimental Setup Window the Start Run tab is displayed The Start Run tab includes a section for checking information about the run that is going to be started including the selected protocol and plate file and a section for selecting the instrument block Check the RUN Information This information includes the protocol name the plate name and optional added notes 2 Click the Start Run button to begin running the experiment on the selected block Calibration Wizard The CFX system is factory calibrated for commonly used fluorophores in white well and clear well plates T
85. ied by the last end cycle Use these data to determine if a specific target sequence is present positive in a sample Positive targets have higher RFU values than the cutoff level you define The software displays these data in the End Point tab Settings Adjust data analysis settings Results Shows the results immediately after you adjust the Settings Well Selector Select the wells with the end point data you want to show Well spreadsheet Shows a spreadsheet of the end RFU collected in the selected wells Va Data Analysis Sample End Point Multiplex Data opd File View Settings Tools S a ra A wae Well Group All Wells vi es End Port FA Run Information Settings p End Fluotophere Os 5 Wel A Flw gt Conten Sample pry Cal 9 End Cycles ToAverage 2 S All Cy5 Neg Ch 1924 RFUs RFUs 803 Cy5 Std 1 2811 Positive O Percent of Range 219 a B04 Cy5 Std 2 3088 Positive 805 Cy5 Std 3 3066 Positive ions B06 Cy5 Std 4 3048 Positive Lowest RFU value 1593 B07 C5 Std 5 3188 Positive Highest RFU value 3784 Bos Cy5 Sid 6 3223 Positive Negative Control Average 1787 B09 C5 Sid 7 3532 Positive f Cut Off Value 2006 i B11 Cy5 Neg Cut 1 1880 10 Wei 12 003 Cy5 Std 1 2820 Positive Neg C04 Cy5 Std 2 3123 Positive C05 Cy5 Std 3 3152 Positive 1 2 c AOA J AIAN ETA E 9 Neg is Hasna RE AE 3147 Positive Coe oe Es nest 007 C5 Sds 3259 Positive
86. ights pane click a box under the name of the role to add or remove that right Click one or more rights in the list To change all the rights for all the roles to the default list click Restore Default Rights Click OK to open a dialog box and confirm that you want to close the window Click Yes to close the dialog box and window To view your current user role and rights select User gt User Administration Contact a software administrator to modify the user settings rights and roles listed in the User Administration window A Principal Operator or Guest user can view only their own user settings rights and roles CFX96 and CFX96 Deep Well Systems Instruction Manual 11 Resources Read this chapter to learn more about resources for the CFX system e LIMS Integration page 99 e Calibration Wizard page 102 e Instrument maintenance page 104 e Application log page 106 e Troubleshooting page 106 e References page 108 LIMS Integration CFX Manager software can be configured for use with a laboratory information management system LIMS For LIMS integration CFX Manager software requires plate setup information generated by the LIMS platform a LIMS file plrn a protocol file created using CFX Manager software prcl a defined data export location and a defined export format Creating a LIMS File A LIMS file plrn contains the plate setup details and the protocol file name CFX Manager software will
87. ine 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 49 to change the default baseline for selected wells To open this window 1 Select Settings gt Baseline Thresholds to open the Baseline Thresholds window To adjust the begin and end baseline cycle for each well 1 In the Baseline Cycles pane select one or more wells by clicking the row number clicking the top left corner to select all wells holding down the Control key to select multiple individual wells or holding down the shift key to select multiple wells in a row 2 Adjust the Baseline Begin cycle and Baseline End cycle for all selected wells or change the Begin and End cycle number at the bottom of the spreadsheet Figure 49 3 Click OK to confirm the change and close the window 59 Data Analysis Overview Select the Analysis Mode Select the Analysis Mode to determine the method of baseline subtraction for all fluorescence traces Select Settings gt Analysis Mode to choose one of these three options e No Baseline Subtraction The software displays the data as relative fluorescence traces Some analysis is not possible in this analysis mode e Baseline Subtracted The software displays the data as baseline subtracted traces for each fluorophore in a well The software must baseline subtract the data to d
88. ing label Table 3 Meaning of safety warning labels CAUTION Biohazard This symbol identifies components that may become contaminated with biohazardous material CAUTION Risk of danger This symbol identifies components that pose a risk of personal injury or damage to the instrument if improperly handled Wherever this symbol appears consult the manual for further information before proceeding CAUTION Hot surface This symbol identifies components that pose a risk of personal injury due to excessive heat if improperly handled Instrument Safety Warnings The warning labels shown in Table 4 also display on the instrument and refer directly to the safe use of the system Table 4 Instrument safety warning labels Icon Meaning Warning about risk of harm to body or equipment Operating the CFX system before reading this manual can constitute a personal injury hazard For safe use do not operate this instrument in any manner unspecified in this manual Only qualified laboratory personnel trained in the safe use of electrical equipment should operate this instrument Always handle all components of the system with care and with clean dry hands Table 4 Instrument safety warning labels continued Icon Meaning Warning about handling biohazardous materials When handling biohazardous samples adhere to the recommended precautions and guidelines and comply with any local guidelines specific to your laboratory and locat
89. ion Warning about risk of burning A thermal cycler generates enough heat to cause serious burns Wear safety goggles or other eye protection at all times during operation Always allow the sample block to return to idle temperature before opening the lid and removing samples Always allow maximum clearance to avoid accidental skin burns Warning about risk of explosion The sample blocks can become hot enough during the course of normal operation to cause liquids to boil and explode Safe Use Specifications and Compliance Table 5 lists the safe use specifications for the CFX system Shielded cables Supplied must be used with this unit to ensure compliance with the Class A FCC limits Table 5 Safe use specifications Safe Use Requirements Specifications Temperature Indoor use Ambient temperature 15 31 C Relative humidity maximum 80 noncondensing Altitude re Up to 2 000 meters above sea level REGULATORY COMPLIANCE This instrument has been tested and found to be in compliance with all applicable requirements of the following safety and electromagnetic standards e IEC 61010 1 2001 2nd Ed EN61010 1 2001 2nd Ed Electrical Equipment For Measurement Control and Laboratory Use Part 1 General Requirements IEC 61010 2 010 2005 EN61010 2 010 2003 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 010 Particular requirements for laboratory equipment for the heatin
90. k Copyright Notice 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 list of conditions and the following disclaimer in the documentation and or other materials provided with the distribution 3 The end user documentation included with the redistribution if any must include the following acknowledgment This product includes software developed by the University of Chicago as Operator of Argonne National Laboratory 108 Bio Rad Laboratories Inc 1000 Alfred Nobel Drive Hercules CA 94547 Bio Rad 3 boulevard Raymond Poincar pec REP 92430 Marnes la Coquette France Tel 33 0 1 47 95 60 00 Fax 33 0 1 47 41 91 33 www bio rad com Bio Rad BIO RAD Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 3065 7550 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 Life Science Gr oup France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Isr
91. l Systems Instruction Manual Data File Reports The Report window Figure 65 shows information about the current data file in the Data Analysis window To open a report select Tools gt Reports or click the Reports button on the toolbar in the Data Analysis window The Report window shows these three sections e Menu and toolbar Select options to format save and print the report or template e Options list top left side of window Select options to show in the report e Options pane bottom left side of window Enter information about a selected option e Preview pane right side of window View the current report in a preview Report 1_FamHexTexCy5 Delete Well perd Fle Terglstes Forma 1_FamHexTexCy5 Delete Well pcrd Pararse 12 12 07 02 54 PM vj Daa 7 Gene Expression Analis Settings Report Information V Chat A Taget Names Experiment Date 5 10 07 11 55 AM V Sample Nares User menn Data File Name 1_FamHexTexCy5 Delete Well perd Dats File Path C Documents and Settings All Users Documents Bio RadiCF X Users edimn Selected Well Group All Wells 1_FamttexT exly5 Delete Wel pord Sub Tae Experiment Setup 12 12 07 02 54 PM Run Information Run User Neel IO Notes Sample Volume 0 Lid Temperature 105 Lid Force Not Available Protocol 1 95 0 C for 3 00 2 95 0 C for 0 10 3 55 0 C for 0 30 Figure 65 Example of a Report window for a data file TIP The layout of the report can de
92. le Name OHr iHr 2Hr OHr 2Hr Or 2Hr Exit Plate Spreadsheet Quanity NJA NJA NJA NJA NJA NJA NJA NJA NJA 100000000 00 10000000 00 1000000 00 100000 00 10000 00 1000 00 100 00 100000000 00 Units copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number copy number v Figure 30 Plate Spreadsheet View window Open the spreadsheet view to import or export the well contents to Excel or to another tab delimited format Click Import Template to import well contents from a comma delimited file Click Export Template to export well contents in Excel file csv format Sort or edit a column by selecting it and using these methods Sort the spreadsheet according to the data in one column by clicking the diamond next to a column name Edit the contents of a column that has an asterisk at top by clicking and typing in each well NOTE Select the units for the standard curve data in the Quantity column by opening the Plate Editor and selecting Settings gt 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 Rig
93. liak tli i tial Ras St fowls 79 OCF 2 a Figure 61 Layout of the End Point analysis tab The Results list includes this information Lowest RFU value Lowest RFU value in 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 cutoff value will be called Positive To adjust the cutoff value change the RFU or Percentage of Range The Cut Off Value is calculated using this formula Cut Off Value Negative Control Average Tolerance Select a tolerance by one of these methods RFUs default Select this method to use an absolute RFU value for the tolerance The minimum RFU tolerance value is 2 The maximum is the absolute value of the highest RFU value minus the absolute value of the lowest RFU value The default RFU tolerance value is 10 of the total RFU range Percent of Range Select this method to use a percentage of the RFU range for the tolerance The minimum percent of range is 1 percent The maximum percent of range is 99 percent The default percent of range is 10 percent 71 Data Analysis Windows Adjusting the End Point Data Analysis Adjust the information shown in the End Point tab by following these methods e Choose a Fluorophore fro
94. listed in Table 21 Table 21 Results spreadsheet content Information Description Well Well in the plate Fluor Fluorophore detected Content Sample type and replicate number Target Amplification target name gene Sample Sample description Threshold Cycle C t Threshold cycle C t Mean Mean of the threshold cycle for 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 denaturation annealing and extension or one round of annealing and extension steps in a protocol CFX96 and CFX96 Deep Well Systems Instruction Manual Plate Spreadsheet Select the Plate spreadsheet to see a plate map of the data for one fluorophore at a time Select each fluorophore by clicking a tab at the bottom of the spreadsheet Figure 58 shows the Plate spreadsheet as plate map ff Quantitation Plate ka 3 Duantitation Data alll Gene Expression 208 End Point Allelic Discrimination Output Content Sample Cit Starting Quantity 1 2 3 4 5 G 7 Content Std Std 2 Std 3 Std 4 Std 5 Std 6 Cit 14 39 1779 21 20 24 49 27 20 28 54 Content Std 1 Std 2 Std 3 Std 4 Std 5 S
95. ls must include the following e Two or more targets The two targets that represent different amplified genes or sequences in your samples e One or more reference targets At least one target must be a reference target for normalized expression Assign all reference targets in the Experiment Settings window page 38 to analyze the data in Normalized Expression mode AAC t Experiments that do not contain a reference must be analyzed using Relative Expression mode AC t e Common samples Your reactions must include common samples minimum of two required to view your data plotted in the Gene Expression tab These samples represent different treatments or conditions for each of your target sequences Assign a control sample optional in the Experiment Settings window page 38 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 66 shows an example of the minimum contents of the wells for a singleplex gene expression experiment Targeti Targeti Sample Sample2 Unk Target2 Target2 Samplei Sample2 Figure 66 Example of well contents in a singleplex gene expression experiment Figure 67 shows an example of the minimum contents of the wells for a multiplex gene expression experiment Targeti Target2 Sample1 Targetl Target2 Sample2
96. m operations displays the date and time the name of the logged in user the system status the type of reaction module and thermal cycler name and any attached S1000 thermal cyclers NOTE The C1000 thermal cycler stores up to 20 real time PCR experiment runs using a date time stamp on the runs When 20 runs are stored on the C1000 older run data is deleted when a new run is stored CFX96 and CFX96 Deep Well Systems Instruction Manual eer ey J000 Series 07 30 2008 11 07 37 Tharmal Cycling Platform i AHER Thermal Cycler Reaction Module Status Ge CCOO3412 LFA a04 Idle Figure 32 Start up screen on the front panel To initiate the functions in the main menu press the associated function keys F1 through F4 e Log In F1 Log in to the C1000 thermal cycler Once you log in the button name changes to Log Off e Files F2 View the files and folders in the file library e Utilities F3 Open the Utilities menu e New Protocol F4 Create a new protocol Creating a New Experiment 1 Select New Protocol F4 in the start up screen to begin Figure 33 None a Vol tid Ec a5 ORS Use the keypad to enter values and navigate Est 01 13 00 through the protocol steps Figure 33 Default real time PCR protocol 2 To change the target temperature and the hold time in a temperature step press the arrow keys to navigate between steps and to select a parameter temperature or time Press the alphanumeric keys
97. m the pull down list to view the data e Choose an End Cycle to Average value to set the number of cycles that the software uses to calculate the average end point RFU e Select RFUs to view the data in relative fluorescence units e Select Percentage of Range to view the data as a percentage of the RFU range Allelic Discrimination Tab 12 The Allelic Discrimination tab assigns the genotypes to wells with unknown samples using the RFU or C t of positive control samples Figure 62 Use this data to identify sample genotypes including Allele 1 Allele 2 Heterozygote Unknown Control 1 or Control 2 NOTE The data for allelic discrimination must come from multiplex experiments Allelic discrimination analysis requires the following minimal well contents e Two fluorophores in each well except the wells that contain positive controls can contain only one fluorophore e One fluorophore that is common to all wells in the well group e NTC no template control samples if you want to normalize the data Data Analysis 2 Target Allelic Discrimination perd Sec File Yiew Settings Tools E S A A Ry viewsedit Plate Ge well croup all wels F 7 Se a Va Quantitation A Quantitation Data alll Gene Expression 298 End Point Allelic Discrimination Kh ac PY Run Information Allelic Discrimination Well Sample 6 Cit C2 9 Call S Type z BO6 30 61 31 53 Allele 1 Auto a COB 30 76 31 83 Allele 1 Auto DOE 29
98. n appropriate three pronged electrical outlet Press the power switch on the back panel of the C1000 thermal cycler to start the system 7 Follow the instructions in the front panel of the C1000 thermal cycler to remove the red shipping screw from the inner heater lid Figure 6 Turn the screw counterclockwise to lift it out of the hole Shipping Screw Status Shipping Screw is inserted 1 Open Optical Module lid press manual button below the Bio Rad logo 2 Remove RED Shipping Screw Pom hole adjacent to left side of well B1 3 Close Optical Module lid press manual button positioned in Font of black 4 Press F1 Screw Removed to confirm Shipping Screw has been removed To checkiremowe the shipping screw status Follow the instructions Figure 6 Instructions to remove the shipping screw NOTE If the shipping screw is not removed at this step it will be detected by CFX Manager software Follow instructions to remove the screw page 15 TIP The shipping screw must be in place when the module is shipped Save this screw in a safe place for future shipping 8 Remove the shipping plate from the thermal cycler block to operate Installing CFX Manager Software CFX Manager software is run on a personal computer PC and is required to analyze data from the CFX system and to control the CFX system in software controlled mode Table 6 lists computer system requirements for CFX Manager software version 1 6 o
99. n pair wise comparison 2 If any target has the same number of common replicate groups then set the dominant calibrator to the target with the smallest range of AC t values in pair wise comparisons The range is examined by comparing the absolute value of the difference between the maximum and minimum AC t for the inter run calibrators of a given target 3 If any target has an identical range as the AC t values then set the dominant calibrator to the target with the smallest absolute value of average AC t for eligible inter run calibrator samples 4 If any target has identical average AC t absolute values then set the dominant calibrator to the replicate group with the smallest AC t NOTE The first data file imported into the Gene Study will always serve as the hub file for pairwise data comparison during inter run calibration Gene Study Window The Gene Study window includes two tabs e Study Setup tab Click this tab to manage the experiments in the Gene Study Adding or removing data files in a Gene Study does not change the original data in that file e Study Analysis tab Click this tab to view the gene expression data for the combined experiments Figure 71 shows the Gene Study window lia Gene Study Gene Study Time Course 3 Files mgxd E Study Setup alll Study Analysis C File Name File Fokder Date Created Well GroupNeme Step Sr a Time Couse2 C Documents and Settingsijowery Desktop JOL GE 8 14 2007 11 36 01 AM
100. n the selected well NOTE This spreadsheet only shows as many as two peaks for each trace To see more peaks click the Melt Curve Data tab page 70 69 Data Analysis Windows Va Quantitation a Quantitation Data Melt Curve e Melt Curve Data alll Gene Expression coe End Point hy Qc by Run Information Melt Peak Ga lt gt gt g 70 80 70 80 Temperature Celsius Temperature Celsius SYBR Peak Type Positive v Step Number 7 v 1 2 3 4 5 6 8 4 10 11 12 Well 4 Fluor 4 Content Sampe pee gt a E a ES e os oe oa TET y r owe ser on ee oe sor Sn a D04 SYBR Std 2 83 50 i o ae oes ome ome e oe en 2 Figure 60 Layout of the Melt Curve tab in the Data Analysis window Adjust the Melt Curve data by any of these methods e Click and drag the threshold bars in the Melt Peak chart to include or exclude peaks in data analysis e Select Positive in the Peak Type pull down menu to show the spreadsheet data for the peaks above the Melt Threshold line or select Negative to view the spreadsheet data for the peaks below the Melt Threshold line Melt Curve Data Tab The Melt Curve Data tab shows the data from the Melt Curve tab in multiple spreadsheets that include all the melt peaks for each trace Select one of four options to show the melt curve data in different spreadsheets e Melt Peaks List all the data including all the melt peaks for each trace e Plat
101. nd to set up a gene expression experiment Click the Clear Replicate button to clear the replicates numbers in the selected wells Click the Clear Wells button to permanently clear all content in the selected wells Experiment Settings Window To open the Experiment Settings window follow one of these options 38 CFX96 and CFX96 Deep Well Systems Instruction Manual e Inthe Plate Editor click the Experiment Settings button e While analyzing data in the Data Analysis window click the Experiment Settings button in the Gene Expression tab Open the Experiment Settings window to view or change the list of Targets and Samples Figure 27 or to set the gene expression analysis sample group to be analyzed if Collection Names have been added to the wells e Targets A list of target names for each PCR reaction such as a genes or sequences of interest Click the Reference column to assign reference genes in an experiment e Samples A list of sample names that indicate the source of the target such as a sample taken at 1 hour 1 hr or taken from a specific individual mouse Click the Control column to assign the control condition for an experiment Figure 27 shows the Targets tab with the analysis settings shown Experiment Settings Targets Samples Select To Full Name Reference Color V Show Chat AutoEfficiency Efficiency Benig GAPDH 96 2 IL1b Tubulin Show Analysis Settings Sample Name Grouping Option Target
102. ning 00 34 43 Lid 105 C Shuttle 45 0 C View Edit Plate Replace Plate Figure 19 The Real time Status tab displays the data during a run 22 CFX96 and CFX96 Deep Well Systems Instruction Manual REPLACING A PLATE FILE During arun replace the plate file by clicking the Replace Plate button Figure 19 in the Real time Status tab Select the new plate file pltd from the list in the windows browser NOTE CFX Manager software checks the scan mode and plate size for the plate file these must match the run settings that were started during the experiment TIP Replacing a plate file is especially useful if you start a run with a Quick Plate file in the Express Load folder Time Status Tab The Time Status tab shows a countdown timer for the current run 23 Running Experiments 24 CFX96 and CFX96 Deep Well Systems Instruction Manual 4 Protocols Read the following chapter for information about creating and editing protocol files Protocol Editor window page 25 Protocol Editor controls page 27 Temperature control mode page 30 Protocol AutoWriter page 30 Protocol Editor Window A protocol instructs the instrument to control the temperature steps lid temperature and other instrument options Open the Protocol Editor window to create a new protocol or to edit the protocol currently selected in the Protocol tab Once a Protocol is created or edited in the Protocol Editor click OK to load
103. nning a protocol written in the Protocol AutoWriter window will always result in a PCR product 31 Protocols 32 CFX96 and CFX96 Deep Well Systems Instruction Manual 5 Plates Read this chapter for information about creating and editing plate files e Plate Editor window page 33 e Select Fluorophores window page 35 e Well loading controls page 36 e Experiment settings window page 38 e Well Groups Manager window page 40 e Plate Spreadsheet View window page 42 Plate Editor Window A plate file contains run parameters such as scan mode and fluorophores and well contents and instructs the instrument about how to analyze the data Open the Plate Editor window to create a new plate or to edit the plate currently selected in the Plate tab Once a plate file is created or edited in the Plate Editor click OK to load the plate file into the Experiment Setup window and run it To run an experiment you must load at least one well with sample type and fluorophore TIP Click the Plate Loading Guide button to open the Plate Loading Guide window from the toolbar for a quick overview of instructions to load a plate Opening the Plate Editor To open the Plate Editor window Figure 25 follow one of these options e To create a new plate select File gt New gt Plate or click the Create New button in the Plate tab page 19 e To open an existing plate select File gt Open gt Plate or click the Open Existing button in
104. normalize the relative differences in a target concentration between samples Typically message levels for one or more reference genes are used to normalize the expression levels of a gene of interest Reference genes take into account loading differences or other variations represented in each sample and they 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 numbers of transfected sequences in a PCR reaction The most common application for gene expression study is the comparison of cDNA concentration in more than one reaction to estimate the levels of steady state messenger RNA The software calculates the relative expression level of a target with one of these scenarios e Relative expression level of a target sequence Target 1 relative to another target Target 2 For example the amount of one gene relative to another gene under the same sample treatment e Relative expression level of one target sequence in one sample compared to the same target under different sample treatments For example the relative amount of one gene relative to itself under different temporal geographical or developmental conditions 19 Gene Expression Analysis Plate Setup for Gene Expression Analysis To perform gene expression analysis the contents of the wel
105. ns of buttons in the toolbar Table 16 Toolbar in the Data Analysis window Toolbar button Function Save Save the current data file Print Print the selected window Trace Style Open Trace Style window A dE Ge CFX96 and CFX96 Deep Well Systems Instruction Manual Table 16 Toolbar in the Data Analysis window continued Toolbar button Name Function Report Open a Report for the current data file View Edit Plate Open the Plate Editor to view and edit the contents of Gd View Edit Plate he wells ed t Well Groups Select a well group name from the pull down menu O At The default selection is All Wells Help Open the software Help site for more information about data analysis Data Analysis Menu Bar Table 17 lists the functions of items in the menu bar Table 17 Menu bar items in Data Analysis window Menu Item File View Settings Tools Save Save the file Save As Save the file with a new name Repeat Experiment Extract the protocol and plate file from the Current experiment to rerun it Exit the Data Analysis window Run Log Open a Run Log window to view the run log of a data file Analysis Mode Select Baseline Subtraction method for the selected well groups in the data C t Determination Mode Select Regression or Single Threshold mode to determine how C t values are calculated for each trace Baseline Thresholds Open the Baseline Thresholds window to adjust the baseline or the thre
106. nt pane and then clicking a button in the Selected Instrument pane Figure 11 Selected Instrument Open Lid Close Lid All Instruments View Summary Figure 11 Buttons at the bottom of the Detected Instruments pane e Click View Status to open the Run Details window to check the status of the selected instrument block e Click Open Lid to open the motorized lids on the selected instrument e Click Close Lid to close the motorized lids on the selected instrument e Click View Summary to open the Instrument Summary window 13 CFX Manager Software If only one instrument is detected then the View Summary button does not appear To view the Instrument Summary window for a single instrument select View gt Instrument Summary Status Bar The left side of the status bar at the bottom of the main software window shows the current status of instruments View the right side of the status bar to see the current user name date and time Click and drag the right corner of the status bar to resize the main window Instrument Properties Window 14 To open the Instrument Properties window to view information about an instrument right click on the instrument icon in the Detected Instruments pane Figure 10 on page 13 Properties Tab The Properties tab displays important serial numbers for the connected instrument including the thermal cycler and reaction module The firmware versions are also displayed The default name
107. nts and in the software and for troubleshooting To open the Application log in the main software window select View gt Application Log Event Log for BioRadPCRClientHistoryL_og xml File BioRadPCRChentHistoryLog xml Find gt Date 9 Message Severity Log a 10 31 2007 9 50 33AM_ Started protocol run run defintion ME THOD Info CFXS6E M00 CALC HOTLID 105 30VOLUME 25 TEMP 10 31 2007 9 50 24 M CFXS6EMO0 Finished synchronization Info CFXS6E MOD 10 31 72007 9 50 24 4M CFXS6EMO1 Finished synchronization Info CFXS6EMO01 10 31 2007 9 50 224M CFXS6EMO1 Started synchronization Info CFXS6E M01 19721 72017 IRN 72 AM CEXORE MON Started cuneheanization Infa CFXARE MNN v lt IKK lt gt S Event Log gt Event Time 2007 09 12T08 49 48 080 07 00 Message BioR adCFXM anager Client Application shutdown Logged in user was BioR ad Grant Trace Figure 83 Example of an Event Log file Troubleshooting Typically software and instrument communication problems can be resolved by restarting your computer and the system Be sure to save any work in progress before restarting NOTE Check that your computer has sufficient RAM and free hard drive space The minimum RAM is 2 GB and the minimum hard drive space is 20 GB Installing the Software Manually If needed install the software manually by following these instructions 1 Insert the software CD 2 Right click the software CD icon and select Explore to open the C
108. ocal Bio Rad representative for country specific instructions Vi CFX96 and CFX96 Deep Well Systems Instruction Manual Table of Contents Chapter 1 System Installation 000 00 ce ee 1 Unpacking the Optical Reaction Module n a anaana aaaea 1 System Requirements na nn nananana eee eens 1 System Overview ret eureen len d e ne a ee a e e a 2 Setting Up the System nanana aana eee ees 3 Installing CFX Manager Software 0 00 ccc eee ee ee eee 5 SOWO ICS me axe ia etees Se oe tek ee eB at a a Ee ee ee 7 RUNNING EXPGHIMGNIS cireres eed ier kee eat A Ew hee eae 7 Chapter 2 CFX Manager Software 0 00c eee e eee eee 9 Main Software WindOW essees sste gigire eta eee eee ees 9 SUED VVIZ AUC sah 5 sy di cee cae a eee ae nen ake a a Re dea So here Se 12 Detected Instruments Pane 00000 ees 12 Instrument Properties WiINdOW 0 0 0 ee eens 14 Chapter 3 Running Experiments 200 cnc eee e eee ees 17 Experiment Setup Window 0 000 c cee eee eens 17 PYOLOCONWAD aca aire o1t cae eed eer a Ae ek eh oh a dee a 18 Plate PAO iS a ease Oe ee eee a a ie ta aid Sea ae en eee atk ae ed 19 Slat RUN Labi raneta i ee Fa acs ee ee ee ee 19 MUM Details VWINGOW crror oe cc dere cots a Bled a ade de be ce ene an line 20 Chapter 4 Protocols iss 2seewae Jeet eis Seatac es ead ae eee tees 25 Protocol Edito WINGOW serar coc bees eee Let eees iP hewd Keehe ees Pies 25 ProtOCOlEdItOrGONIOIS s
109. ocol and other information about the run for each experiment You can also enter and edit the run Notes by typing in the Notes box or enter and edit the data ID for the run by typing in the ID box Quantitation Quantitation Data Melt Curve Melt Curve Data g Gene Expression 200 End Point Zh ac Protocol 2stepstandard50melt0 Sinc prel Description Exclude 9 Value Us O Results 4 Wells from Analysis Negative control with a Cft less than 38 m NTC with a Clt less than 38 NAT with a Cit less than 38 Positive control with a C t greater than 30 Unknown without a Cit p Standard without a Cft p Efficiency greater than 110 0 Efficiency less than 90 0 Std Curve R 2 less than 0 980 Replicate group C t Std Dey greater than 0 2 p NTC with a C t less than 38 No wells fail this QC Rule Figure 63 QC tab layout 1 950 C for 010 3 00 0 30 Plate Read pP 2 0Co 3 550C for GOTO 2 49 more times 95 0 C for 1 00 1 00 4 5 6 550 C for 7 Melt Curve for 0 05 55 0 to 95 0 C increment Plate Read 0 5 C END Run Information Notes Created By User admin Run Started 12 18 2007 2 55 47 PM Sample Yol Lid Temp 25 105 CFX Serial Number EP123 Base Serial Number CC001055 CFX Manager Version 1 0 963 1218 Figure 64 Layout of the Run Information tab in the Data Analysis window 74 CFX96 and CFX96 Deep Wel
110. odule extends 2 inches over the edge of the desk as shown in Figure 84 Figure 84 Setting up the Optical Module to remove the locking plate 3 With an Allen wrench remove the two large screws from under the front edge of the reaction module below the button for opening the lid Do not remove the two small screws that are located at the front edge of the module You should hear the locking latch release from inside the module Figure 85 shows the two large screws Figure 85 Remove these screws to unlock the optical module 107 Resources 4 Push the reaction module lid open Notice that the latch dark plastic is no longer attached Remove your samples from the block 5 Reassemble the reaction module with the lid open by replacing the locking latch and securing it with the large screws Figure 86 shows the locking latch in place Figure 86 Optical module locking latch References Breslauer Kd et al 1986 Predicting DNA duplex stability from the base sequence Proc Nat Acad Sci 83 3746 50 Livak JL et al 1995 Towards fully automated genome wide polymorphism screening Nature Genetics 9 341 342 Pfaffl MW 2001 A new mathematical model for relative quantification in real time RT PCR Nucleic Acids Research 29 9 2002 2007 Vandesompele J et al 2002 Accurate normalization of real time quantitative RT PCR data by geometric averaging of multiple internal control genes Genome Biology 3 7 1 12 Minpac
111. older button RUN Figure 36 Entering a protocol name 4 Click Run F2 to continue and run the protocol Figure 37 FILE MANAGEMENT File Library File USERS ACMINVRUN a METHOD CALC WAOTUD 95 30 ly PREVIOUS RUNS Flay SYSTEM Flt LISERS Si ADMIN Figure 37 Protocol successfully saved 47 Stand Alone Operation 5 Edit the Sample Volume and Lid Temperature A Sample ID or User can also be recorded for the run Figure 38 Click OK F1 to proceed Run RUNI on CLOOS412 Sample volume pi Lid Temperature lg alt i ap F Turn on hotlid Confirm settings and press RUM of OE to proceed a ee eee Figure 38 Editing sample volume and lid temperature 6 Select a Scan Mode to collect fluorescence data during a run Figure 39 Running real time protocol Scan Mode O All Channels FRET Data File Mame zpecr 000r a0 111150 _Ccous4l2 RTRUA Select real time run mode Figure 39 Scan mode and data file name Scan modes detect calibrated fluorophores in these channels e All Channels Collects data from channels 1 through 5 on the CFX96 and CFX96 Deep Well systems e SYBR FAM Collects data only from channel 1 and provides a fast scan e FRET Collects data only from the FRET channel and provides a fast scan 7 A default stand alone data file name is created prior to the run If you wish to change the name use the arrow keys to navigat
112. olds The software takes an average C t or RFU for the positive controls to automatically set the threshold lines for discrimination of the alleles Adjust the positions of the threshold bars by clicking and dragging them and the software automatically adjusts the calculations to make new genotype assignments e f the experiment contains three controls in the plate then the positions of the threshold bars are based on the mean and the standard deviation of the RFU or C t of the controls e fthe number of controls is less than three then the positions of the threshold bars is determined by the range of RFU or threshold cycle values in the selected fluorophore Adjust allelic discrimination data by following any of these methods e Click and drag the threshold bars in the Allelic Discrimination chart to adjust the calls in the spreadsheet e Select a fluorophore for each axis in the chart X and Y in the settings options on the bottom right of the window e Change a call manually by highlighting a row in the spreadsheet and then selecting an option in the Call Selected Alleles list including Allele 1 Allele 2 Heterozygote None Unknown Control 1 or Control 2 e Click the Restore Default Thresholds button to restore the vertical and horizontal bars to their original positions which are indicated by the numbers next to the bars e Select the C t Display Mode to view the data as threshold levels Select RFU Display Mode to view the d
113. oncentration box To delete a concentration select it press the Back Space key on your keyboard and then press Enter Select multiple wells with a Standard sample type to activate the Dilution Series button Click the Dilution Series button to enter a dilution series for the concentration of Standard samples and load a standard curve Enter the Starting Concentration for the dilution series the Replicates from starting replicate number and to ending replicate number and the Dilution Factor amount to change the concentration with each replicate group Select Increasing for a dilution series that increases or select Decreasing for a dilution series that decreases Finally select the fluorophore used for the dilution series from the pull down menu and click Apply Select Tools gt 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 68 Select Tools gt Show Collection Name to show this pane Enter sample collection information about one or more wells by selecting the wells and typing a collection name in the pull down menu Any collection name you add to wells appear in the Gene Expression Analysis window and enables sample grouping options Click the Experiment Settings button to open the Experiment Settings window to manage the lists of Targets and Samples a
114. ot edit Predefined 5 Plate Type Enter BR White BR Clear or Required other calibrated plate type B Scan Mode Enter SYBR FAM Only All Required Channels or FRET B 7 Units Enter one of the following copy Required number fold dilution micromoles nanomoles picomoles femtomoles attomoles milligrams micrograms nanograms picograms eC aes attograms or percent B Run ID Enter short description or Optional barcode identifying this run Note Do not use commas in run ID B Run Note Enter run description Note Do Optional not use commas in run ID B Run Protocol Protocol file name protocol file Required and plrn file must exist in same folder A 11 amp 12 Data File TBD Do not edit Predefined A Plate Data Do not edit Predefined A 14 110 Well Position Do not edit Predefined CFX96 and CFX96 Deep Well Systems Instruction Manual B G 14 110 Ch1 Dye Ch2 Enter one calibrated dye name Required Dye Ch3 Dye for Ch4 Dye Ch5 example FAM for each Dye FRET channel being used H Sample Type Enter one of the following sample Required types Unknown Standard Positive Control Negative Control NTC or NIRT J O 14 110 CH1 Target Enter target name for each Optional CH2 Target channel CH3 Target used CH4 Target CH5 Target FRET Target P Biological Set Enter biolog
115. pression Qc PCR Quantitation Analysis Mode Baseline Subtracted Curve Fit C t Determination Mode Regression Single Threshold LogViews O On of Allelic Discrimination Display Mode Threshold Cycle RFU No End Point End Cycles to vg PCR 5 4 End Point Only Run 2 4 Figure 78 Data Analysis tab in the User Preferences window For the quantitation data select the following settings e Analysis Mode Select the default base lining method for the analysis mode Choose Baseline Subtracted Curve Fit No Baseline Subtraction or Baseline Subtracted e C t Determination Mode Select between Regression mode or Single Threshold mode to determine how C t values are calculated for each fluorescence trace e Log View Select On to show a semi logarithmic graph of the amplification data Select Off to show a linear graph For the allelic discrimination data select the following settings e Display Mode Select RFU to show the data as a graph of the RFU or select Threshold Cycle to show a graph of threshold cycles e Normalize Data This selection is only available when RFU is selected Select No to show non normalized data Select Yes to normalize the data to the control sample For end point data select the following settings Select the number of end cycles to average when calculating the end point calculations e PCR Enter a number of cycles for PCR to average the end cycles for quantitation data default is 5 e
116. r higher Table 6 Computer requirements for CFX Manager software version 1 6 or higher System Recommended Operating system Windows 7 and Windows 8 Windows 7 or Windows 8 Drive Hard drive CD ROM drive CD RW drive 10 GB 20 GB Processor speed 2 0 GHz 2 0 GHz RAM 2 GB RAM 2 GB RAM Screen resolution 1 024 x 768 with true color mode 1280 x 1024 with true color mode USB USB 2 0 Hi Speed port USB 2 0 Hi Speed port To install CFX Manager software 1 Make sure you are logged in with administrative privileges The software must be installed on the computer by a user with administrative privileges System Installation 2 3 4 5 Place the CFX Manager software CD in the computer s CD drive The software launch page should appear automatically Double click Install Software on the software launch page Figure 7 id x Install Software Documentation Firmware Drivers PCR Legal and Authorization Notices Figure 7 Software installation screen Follow the instructions on screen to complete installation When completed the Bio Rad CFX manager software icon will appear on the desktop of the computer If the launch page does not appear automatically double click on CD drive Bio Rad CFX then open and follow instructions in the Readme txt file See Installing the Software Manually on page 103 Installing the Drivers If the CFX system is going to be run in Software controlled mode
117. rected standard deviation of the unscaled expression Expression SD Expression Relative expression Wells Well number in the plate Gene Study Report Window Open the Gene Study Report window to arrange the Gene Study data into a report To create a gene study report follow these steps 90 l 2 3 Adjust the Gene Study report data and charts as needed before creating a report Select Tools gt Reports to open the Gene Study report window Click the check boxes in the report options list to select and remove options to choose the data to display Click the Update Report button to update the report preview pane The report preview pane shows a view of the report Print or save the report Click the Print button in the toolbar to print the current report Select File gt Save to save the report as a PDF Adobe Acrobat Reader file MHT Microsoft document or MHTML Microsoft document formatted file and select a location to store the file Select File gt Save As to save the report with a new name or ina new location CFX96 and CFX96 Deep Well Systems Instruction Manual 10 Users and Preferences Read this chapter to learn more about managing software users and their preferences e Log In or Select User page 91 e User Preferences window page 92 e Configure email notification page 93 e User Administration page 97 Log In or Select User CFX Manager software manages multiple users and their preferences The
118. res the data file for each experiment in the Gene Study Date Created Date the run data were collected Well Group Name Name of the well group that was selected when the file was added to the Gene Study TIP To analyze one well group in the Gene Study that well group must be selected in the Data Analysis window before importing the data file into the Gene Study Step Protocol step that included the plate read to collect real time PCR data Grid View Open a plate map of the plate with the data in each of the experiments included in the Gene Study Study Analysis Tab The Study Analysis tab shows the data from all experiments that are added to the Gene Study Open this tab to analyze the data and select these options for the Gene Expression chart Mode Select Normalized Expression AAC t or Relative Quantity AC t Graph Data Select Relative to normal or Relative to control in the graph x axis options Select the labels on the x axis of the graph including Sample or Target y axis options Change the labels on the y axis of the graph including Linear Log 2 or Log 10 Scaling Options Choose Highest value Lowest value or leave the data Unscaled This option is only available when your samples do not contain controls CFX96 and CFX96 Deep Well Systems Instruction Manual e Graph Error Select the multiplier for standard deviation bars in the graph including 1 2 or3 e Experiment Settings button Choose the show options for targ
119. rmation required to conduct a LIMS compatible run Gene Study View and analyze in Gene Study window Stand alone pre data Contains fluorescence readings from stand alone file operation that is converted into a data file Running Experiments Recommended Plastic Consumables The CFX system accepts low profile 0 2 ml plates and tubes Bio Rad recommends the following consumables for optimal results e MLL 9601 Low profile 96 well unskirted plates with clear wells e MLL 9651 Low profile 96 well unskirted plates with white wells e HSP 9601 Hard Shell 96 well skirted plates with white shell and clear wells e HSP 9655 Hard Shell 96 well skirted plates with white shell and white wells e TLS 0801 Low profile 0 2 ml 8 tube strips without caps clear wells e TLS 0851 Low profile 0 2 ml 8 tube strips without caps white wells e TCS 0803 Optical flat 8 cap strips for 0 2 ml tubes and plates NOTE High profile plates can also be used with the CFX96 Deep Well system Plate Seals e MSB 1001 Microseal B adhesive seals optically clear strong adhesive based e MSC 1001 Microseal C optical seals optically clear pressure activated adhesivebased Loading the Block To load your reactions in the block follow these suggestions e Click the Open Lid button located on software s Start Run tab see Start Run Tab on page 19 or press the lid button on the front of the system Figure 1 to start opening the motorized lid
120. roup C t Std Dev greater than XX Input a cutoff standard deviation that is calculated for each replicate group User Administration Open the User Administration window in the main software window e Select Users gt User Administration e Click the User Administration button in the menu bar If you log in as an Administrator open the User Administration window to manage users and user rights e Manage Users Add or remove Users and assign each user a Role e 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 80 In this example the Guest user is given the right to save files User Administration f Manage Users Role Password Administrator Principal Manage Rights Managed by Admin strator only Principal Operator Guest Start pause and abort runs Add repeats to a run Perform skip cycles Perform instrument calibration Apply different calibrations to 4 data file Rename instruments Ovenide default lid force settings Restore Default Rights CPCS CON CHSCs caics onanan ooooooos Figure 80 User Administration window with three users Adding and Removing Software Users Only a software Administrator can add and remove users To add software users in the Manage Users pane follow these steps
121. ruse sar ga a ae A ace at sium dae aia E A S 27 Temperature Control Mode anaana anaana aa 30 PrOtOCO AVIOWTIIE cre d kate ee ne aa a AE a 30 Chapter 5 Plates 2ces cesieetawwnees indie aa sacs 33 Prate EARO WINGOW sutersc 5 ece Sa ar tno urtid eRe oe dk Sele te Re al hale WB eaaa 33 Select Fluorophores Window 000 cee ee eee eee 35 Well Loading Control a sande bee dha ees a ee eng ea de wedge is 36 Experiment Settings Window 0 00 cece eee eee eee eee 38 Well Groups Manager Window 0 00 cc eee eee ee ees 40 Plate Spreadsheet View WindoOW 2 0 ee eee eee 42 Chapter 6 Stand Alone Operation 00000 eee eee es 43 GLOOO Thermal Cy Claro encerran ie bead cee a Aad oe ed ork St ales 43 Creating a New Experiment 0000 cece eee eee ee 45 Exporting Data Tor Analysis oui one a Y eN ate Oe ee WS Ee eR 49 Creating DataiFilert s lt roi oe oy odes ke e eed E Seek Bee ers ew ae 51 Vil Chapter 7 Data Analysis Overview 0000 cee eee es 53 Data Analysis Precautions 000 eens 53 Data AnalysiS WINGOW cea de thee eA hee EN ae Ce bade SESE N SH Os 54 QUENULAHON Ido e645 6 ena tere rhe GA Pa eh ee eee ehh S Raed ee eee Rs 57 Data Analysis SettingS eice irk he ee a he ew ee ele ee oe 58 Well SClOClOrS 26 6sc02 aye tien bse Te eG e races be eae E ek eas 60 VALS ris Sst ts heh ais iagety pred eae ok ee ee aoe mr Pessoa a a Rade ee I Bee 62 SPreddsSneelSuw soil eee wh ae a a
122. s to determine C t values e Regression This mode applies a multivariable nonlinear regression model to individual well traces and then uses this model to compute an optimal C t value e Single Threshold This mode uses a single threshold value to calculate the C t value based on the threshold crossing point of individual fluorescence traces CFX96 and CFX96 Deep Well Systems Instruction Manual Adjusting the Threshold In Single Threshold mode adjust the threshold for a fluorophore by clicking on the threshold line in the Amplification chart and moving the mouse pointer vertically Alternatively specify an exact crossing threshold for the selected fluorophore by following these instructions 1 Select Settings gt Baseline Thresholds in the menu bar to open the Baseline Thresholds window 2 Adjust the crossing threshold Figure 49 for the fluorophore by clicking User Defined and entering a threshold number Baseline Thresholds Baseline Cycles Auto Calculated User Defined Bold indicates a changed value aj Fer ol Basse o e oE FAM FAM FAM FAM FAM F amp M FAM FAM FAM FAM FAM FAM FAM AllSelected Rows Begin 2 11 15 18 22 25 26 25 11 15 i won o 2 lt gt 2 2 2 2 2 2 2 2 2 2 2 2 2 R Crossing Threshold Auto Calculated 1151 65 User Defined 1151 65 5 Figure 49 Baseline Thresholds window 3 Click OK to confirm the change and close the window Basel
123. sheets Item Copy Copy as Image Print Print Selection Export to Excel Export to Text Export to XML Export to HTML Find Sort Function Copy the contents of the selected wells to a clipboard then paste the contents into a spreadsheet such as Excel Copy the spreadsheet view as an image file and paste it into a file that accepts an image file such as text image or spreadsheet files Print the current view Print the current selection Export the data to an Excel spreadsheet Export the data to a text editor Export the data to an XML file Export the data to an HRML file Search for text Sort the data in up to three columns 63 Data Analysis Overview 64 CFX96 and CFX96 Deep Well Systems Instruction Manual 8 Data Analysis Windows Read this chapter for more information about the tabs in the Data Analysis window Quantitation tab page 65 Quantitation Data tab page 68 Melt Curve tab page 69 Melt Curve Data tab page 70 End Point tab page 70 Allelic Discrimination tab page 72 QC tab page 74 Run Information tab page 74 Data file reports page 75 Quantitation Tab Use the data in the Quantitation tab Figure 46 on page 57 to set the data analysis conditions including 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
124. shold 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 off then hold down the Control key to temporarily turn on the highlighting Display Threshold Values Display the value of the threshold line in the chart Open the Report for this data file 55 56 Data Analysis Overview Table 17 Menu bar items in Data Analysis window continued Menu Item Command Function Import Fluorophore Calibration Select a calibration file to apply to the current data file Replace Plate Replace the current plate file in the data analysis Export All Data Sheets to Excel Export all the spreadsheet views from every tab to a separate Excel formatted file Help Open software Help for more information about data analysis CFX96 and CFX96 Deep Well Systems Instruction Manual Quantitation Tab Each tab in the Data Analysis window displays data in charts and spreadsheets for a specific analysis method with a well selector to select the data you want to show The Data Analysis window opens with the Quantitation tab Figure 46 in front The Amplification chart data in this tab should be used to determine the appropriate analysis settings for the experiment NOTE The Amplification chart shows the relative fluorescence RFU for each well at ever
125. st recently viewed data files and select one to open in Data Analysis Repeat an Open the Experiment Setup window with the protocol Experiment and plate from a completed run to quickly repeat the run Exit the software program View Application Log Display the application log for the software Run Reports Select a run report to review from a list Startup Wizard Open the Startup Wizard Experiment Setup Open the Experiment Setup window Open the Instrument Summary window Summary Show or hide the Detected Instruments pane Instruments Toolbar Show or hide the main software window toolbar Status Bar Show or hide the main software window status bar User Open the Select User window to change software users Change Password Change your user password Open the User Preferences window User Manage users in the User Administration window Administration Tools Dye Calibration Open the Dye Calibration window to calibrate an Wizard instrument for a new fluorophore Protocol Open the Protocol AutoWriter window to create a new AutoWriter protocol Ta Calculator Open the Ta Calculator window to calculate the annealing temperature of primers View Block Status View a log of the thermal cycler block Log Application Data Open the Application Data folder to view software files Folder User Data Folder Open the Data folder to view protocol plate and data files Properties All View properties of all detected instruments including Instrum
126. t of the names Sample Names Chart of the names Spreadsheet listing the data in each well Gene Expression Melt Curve CFX96 and CFX96 Deep Well Systems Instruction Manual Table 22 Data analysis report categories in the options list continued Category Allelic Discrimination End Point Description Analysis Settings Includes the melt step number and threshold bar setting Melt Curve Chart Copy of the melt curve chart Melt Peak Chart Copy of the melt peak chart Spreadsheet listing the data in each well Analysis Settings Includes display mode fluorophores cycle thresholds and normalized data Copy of the allelic discrimination chart Spreadsheet listing the data in each well Analysis Settings Includes fluorophore end cycles to average mode lowest RFU value highest RFU value and cutoff value Spreadsheet listing the data in each well 1 Data Analysis Windows 78 CFX96 and CFX96 Deep Well Systems Instruction Manual 9 Gene Expression Analysis Read this chapter for information about performing Gene Expression Analysis e Gene Expression page 79 e Plate setup for gene expression analysis page 80 e Gene Expression tab page 80 e Experiment Settings window page 85 e Gene Study page 86 e Gene Study Data spreadsheet page 89 e Gene Study Report window page 90 Gene Expression With the use of stringently qualified controls in your reactions you can run a gene expression experiment to
127. t the experiment before the run e Open a Data File page 53 Open a data file to analyze results e Open a Gene Study page 86 Open a multifile gene expression study to analyze results from multiple gene expression experiments e Open User Preferences page 92 Open the User Preferences window to customize software settings CFX96 and CFX96 Deep Well Systems Instruction Manual Detected Instruments Pane Connected instruments appear in the Detected Instruments pane Figure 10 This list shows each instrument as an icon named with the serial number default Detected Instruments eae e C48FSIM00 B Sa SOGFSIMOT GEX CFK3845IM03 GEX CRXOBSIMO2 Figure 10 Instruments listed in the Detected Instruments pane Right click on the instrument icon or block to select one of these options e View Status Open the Run Details window to check the status of the selected instrument block e Flash Block Indicator Flash the indicator LED on the instrument e Open Lid Open a motorized lid on the selected instrument block e Close Lid Close a motorized lid on the selected instrument block e Rename Change the name of the instrument e Properties Open the Instrument Properties window e Collapse All Collapse the list of instruments in the Detected Instruments pane e 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 Instrume
128. td 6 BRS Content Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Ct 14 25 17 68 21 07 24 25 26 85 20 41 Figure 58 Plate spreadsheet in Quantitation Data tab RFU Spreadsheet Select the RFU spreadsheet to see the RFU readings for each well acquired at each cycle of the experiment Select individual fluorophores by clicking a tab at the bottom of the spreadsheet The well number appears at the top of each column and the cycle number appears to the left of each row Figure 59 Va Quantitation Ei Quantitation Data alll Gene Expression loas End Paint Allelic Discrimination Qc v Step Number 3 A AZ A3 Ad AB AB AP B2 B3 B4 B5 BB 681 231 123 295 6579 106 205 676 289 140 6 27 577 125 396 O822 622 526 628 123 1 22 755 z7 409 328 184 0687 505 813 507 310 118 450 59 273 0204 1 27 0473 302 4260 270 557 242 1 25 26 2 64 0 220 4 31 1 42 1 91 1 63 0 275 5 51 2 4 1 60 0 366 2 3 4 5 Figure 59 RFU spreadsheet in the Quantitation Data tab Melt Curve Tab Open the Melt Curve tab Figure 60 to determine the melting temperature Tm of amplified PCR products This tab shows the melt curve data in these four views e Melt Curve View the real time data for each fluorophore as RFUs per temperature for each well e Melt Peak View the negative regression of the RFU data per temperature for each well e Well Selector Select wells to show or hide the data e Peak spreadsheet View a spreadsheet of the data collected i
129. ter window with a new protocol Creating a Protocol With the Protocol AutoWriter The Protocol AutoWriter window uses information about your reaction to automatically generate a protocol file Follow these steps to use the Protocol AutoWriter to create a new protocol 1 Click the Protocol AutoWriter button on the toolbar to open the Protocol AutoWriter window 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 see Breslauer et al 1986 Select an enzyme type from the list of options iTaq iProof or Other Add parameters in the Additional Parameters Optional pane if you want to add a Gradient Range Hot Start Activation temperature or Final Extension time in the protocol Select a protocol speed Standard Fast or Ultrafast by moving the sliding bar in the Type pane When you move the sliding bar the software adjusts the total run time Select Real time PCR to tell the software to collect fluorescence data Review the protocol in the Preview pane and total run time Make changes as needed Click OK to save the new protocol or click Cancel to close the window without saving the protocol NOTE Bio Rad Laboratories does not guarantee that ru
130. ternatively type a previously determined efficiency To adjust the settings for a sample in the Samples tab e Click a color in the Color column to change the color of the samples graphed in the Gene Expression chart e 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 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 e Target vs Sample e Target vs Collection e Target vs Sample_Collection e Target vs Collection_Sample Well Groups Manager Window 40 Well groups divide a single plate into subsets of wells that can be analyzed independently in the Data Analysis window Once well groups are set up select one in the Data Analysis window to analyze the data in an independent group For example set up well groups to analyze multiple experiments run in one plate or to analyze each well group with a different standard curve NOTE The default well group is All Wells Create Well Groups To create well groups in the Well Groups Manager window follow these instructions 1 Click the Well Groups button in the toolbar of the Plate Editor 2 Click Add to create a new group The pull down menu shows the group name as Group 1 for the first group
131. the assay or plate matches that of the sample Confirm that the appropriate assay was run using the appropriate protocol Check that the controls were included in the assay and generated the expected result Confirm that the assay was run to completion Check that the appropriately diluted samples were loaded into the correct wells and assigned the correct dye Be sure that the samples were run in duplicate at a minimum Check to optimize an examination procedure in order to improve its performance characteristics Care should be taken to note if any variable in the protocol was modified as this could alter the call for the assay These variables can include changes due to e Manually adjusting the baseline e Manually adjusting the threshold e Cq determination mode e Removal of outliers e Removal or changes to QC check rules 53 Data Analysis Overview Data Analysis Window 54 During data analysis changing the way the data are displayed by changing the contents of wells in the Plate Editor never changes the fluorescence data that were collected from each well during the run Once the module collects fluorescence data you cannot delete those data but you can choose to remove data from view and analysis To change the content of wells after a run open the Plate Editor by clicking 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
132. the sample reaches the target temperature in a protocol Enter a sample volume in the protocol editor to select a temperature control mode e Calculated mode When you enter a sample volume between 1 and 50 ul the thermal cycler calculates the sample temperature based on volume This is the standard mode e Block mode When you enter a sample volume of zero 0 ul the thermal cycler records the sample temperature as the same as the measured block temperature Protocol AutoWriter Open the Protocol AutoWriter to quickly write protocols for PCR and real time PCR experiments To open the Protocol AutoWriter select one of these options e Click the Protocol AutoWriter button in the main software window toolbar e Select Tools gt Protocol AutoWriter from the menu bar in the main software window 30 CFX96 and CFX96 Deep Well Systems Instruction Manual Figure 24 shows a protocol bottom of the window written by the Protocol AutoWriter Protocol AutoWriter Erter Target Values Enzyene Additional Parameters Optional Amplicon Length 100 bp Ta Calculator Gradert Range Hot Start Actrvation Annealing Temperature Ta 6 amp 0 S T iTaq Proof Other Final Extension Enter Annealing temperature or use the Ts Calculator The Annealing temperature vall be automaticaly adjusted based on enzyme and speed selections If using Proof 3 C wil be added to the Ta Type Realtime PCR PCR Run Time Figure 24 Protocol AutoWri
133. the units used to describe the concentration of the starting template for wells that contain standards e Scientific Notation Select scientific notation to view concentration units in that notation e Scan Mode Select a default scan mode e Fluorophores Click check boxes to select the default fluorophores that appear in the Plate Editor well loading controls e Libraries Enter the target and sample names used in your experiments These names appear in the lists in the Targets tab and Samples tab in the Experiment Settings window User Preferences mai les Z Frotoco HH Fi ata Analysis ene Expression Pa Email 3 Files Ag P la Data Analysis Gene E Ih ac Settings Plate Type BR White Fluorophores Plate Size 384 wells FAM O ROX C Quase SBR Texas Red C IC Units copy number Fi HE Fi Cal Red 610 Fi User TET C Cys User Scientific Notation C Cal Gold 540 C Quasar 670 C crs Scan Mode All Channels lt gt Libraries Target Hames Sample Hamez Actin OHr GAPDH 1Hr Hr Use text boxes to enter additional names one name per line Figure 77 Plate tab in the User Preferences window CFX96 and CFX96 Deep Well Systems Instruction Manual 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 User Preferences Email 3 Files a Protocol 55 Plate Fa Data Analysis alll Gene Ex
134. tings window to view or change analysis parameters applied in the Gene Expression tab Click a cell in the Color column to change the color of the targets graphed in the Gene Expression chart 85 Gene Expression Analysis e Enter a number for the efficiency of a target The software will calculate the relative efficiency for a target using Auto Efficiency if the data for a target includes a standard curve Alternatively type a previously determined efficiency Figure 70 shows the efficiency of all the targets which appears if Auto Efficiency is selected Experiment Settings Targets Samples Select To Name Full Name Reference Color V Show Chat Auto Efficiency Efficiency E Actin Actin 96 4 GAPDH GAPDH 96 2 ILib ILib 96 8 Tubulin Tubulin 95 0 Show Analysis Settings Sample Name Grouping Option Target vs Sample Figure 70 Targets tab in Experiment Settings window with Analysis Settings selected To adjust the settings for a sample in the Samples tab e Click a color in the Color column to change the color of the samples graphed in the Gene Expression chart e Click a box in the Show Chart column to show the sample in the Gene Expression chart using a color that is selected in the Color column Gene Study 86 Create a Gene Study to compare gene expression data from one or more real time PCR experiments using an inter run calibrator to normalize data between the experiments Create a
135. tion provided by the instrument may be impaired Bio Rad Laboratories Inc is not liable for any injury or damage caused by the use of this equipment in any unspecified manner or by modifications to the instrument not performed by Bio Rad or authorized agent Service of the system should be performed only by Bio Rad personnel Biohazards The CFX system is a laboratory product However if biohazardous samples are present adhere to the following guidelines and comply with any local guidelines specific to your laboratory and location GENERAL PRECAUTIONS e Always wear laboratory gloves coats and safety glasses with side shields or goggles e Keep your hands away from your mouth nose and eyes e Completely protect any cut or abrasion before working with potentially infectious materials e Wash your hands thoroughly with soap and water after working with any potentially infectious material before leaving the laboratory e Remove wristwatches and jewelry before working at the bench e Store all infectious or potentially infectious material in unbreakable leak proof containers e Before leaving the laboratory remove protective clothing e Do not use a gloved hand to write answer the telephone turn on a light switch or touch anything that other people may touch without gloves e Change gloves frequently Remove gloves immediately when they are visibly contaminated e Do not expose materials that cannot be properly decontaminated to potentially in
136. 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 3 Optional To insert a new step select the Insert F1 button To delete a step select the Delete F3 button Figure 33 45 Stand Alone Operation 4 Optional To change step options select the Options F4 button Figure 33 In the Step Options window select a parameter to change including the temperature and time of the step or add remove a plate read to the step Figure 34 t m Step Options Step 1 Gradient d Plate Read A Temperature ga B Gradient Range aT Z Increment Cicycle D Ramp Rate e Cisec E Time seciecycle F 3 Extend secicycle j Figure 34 Step Options window NOTE Press the alphanumeric keys to enter a Gradient Range spanning from 1 to 24 C 5 The GOTO step instructs the thermal cycler to repeat a set of steps in a loop to create the cycles in the PCR experiment Select a GOTO step press the arrow keys to select and then edit the step number in a GOTO step or to change the number of repeats 6 Optional To change the default sample volume select the sample volume box Vol Figure 33 on page 45 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 7 Optional To change the default lid temperature select the lid tempera
137. to select the x axis data of the Gene Expression graph e Target Select this option to graph the target names on the x axis e Sample Select this option to graph the sample names on the x axis Y AXIS OPTIONS The y axis option allows you to show the Gene Expression graph in one of three scales e Linear Select this option to show a linear scale e Log 2 Select this option to evaluate samples across a large dynamic range e Log 10 Select this option to evaluate samples across a very large dynamic range SCALING OPTIONS Select Normalized Gene Expression AAC t to activate the scaling options in the Gene Expression graph Select one of these scaling options to calculate and present your data in a manner that best suits your experimental design e Unscaled expression This option presents the unscaled normalized gene expression e Highest expression Scale the normalized gene expression to the highest for each target by dividing the expression level of each sample by the highest level of expression in all the samples This scaling option uses the scaled to highest formula CFX96 and CFX96 Deep Well Systems Instruction Manual e Lowest expression Recalculate the normalized gene expression for each target by dividing the expression level of each sample by the lowest level of expression in all the samples This scaling options uses the scaled to lowest formula ERROR TYPE Select an option for the type of error calculations error bars in the
138. ture box Lid by pressing the arrow keys Figure 33 on page 45 Use the alphanumeric keys to enter a new temperature The default lid temperatures for the CFX96 modules is105 C NOTE Heating the lid prevents condensation in the sealed reaction vessels 8 When creating a new protocol you have the option to save it with a name Use the arrow keys to navigate to the Protocol Name box and then press the alphanumeric keys multiple times to enter a letter or number to type a new protocol name 9 Press ENTER to accept the name Running the Protocol 1 To begin the run click Done F2 in the Protocol window Figure 33 on page 45 TIP Alternatively click the RUN command key to start the run without saving or editing the name of the protocol 46 CFX96 and CFX96 Deep Well Systems Instruction Manual 2 Enter a protocol name if you have already not done so or edit the name previously created in the Protocol window Use arrow keys to select a destination folder Figure 35 GAVE File Library Folder USERS ADMIN Hee COO lG Fei USERS 7 OR ZAMP SERVICE H PETE A PETER To edit fle name press the Edit Filename button Figure 35 Saving a protocol 3 Click Edit Filename F1 Type a new name in the box and click Save F2 Figure 36 SAYE File Library Folder USERS ADMIN 2 CCOOS 16 j USERS pomm I ZAMP of SERVICE 5 PETE To change Folder press the Change F
139. u in the chart controls of the Gene Expression tab to run a Relative Quantity AC t analysis By definition relative quantity AC t data are not normalized This method is used to quantitate samples that do not include any reference genes targets Typically the researcher is confident in one of the following considerations when setting up the experiment e Each sample represents the same amount of template in each biological sample possibly the same mass of RNA or cDNA in each well e Any variance in the amount of biological sample loaded will be normalized after the run by some method in the data analysis outside of the software For example a researcher might choose to divide the relative quantity value by the normalizing factor possibly the mass of nucleic acid loaded for each sample or the number of cells from which the nucleic acid was isolated Adjusting Gene Expression Data After selecting 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 two options e Relative to control Graph the data with the axis scaled from O0 to 1 If you assign a control in your experiment select this option to quickly visualize upregulation and downregulation of the target e Relative to zero Graph the data with the origin at zero X AXIS OPTIONS The x axis option allows you
140. vs Sample Figure 27 Targets tab in Experiment Settings window Figure 28 shows the Sample Tab with the analysis settings shown Experiment Settings Targets Samples Select To Full Name Control Color Y Show Chart pee mouse 1 mouse 1 Show Analysis Settings Sample Name Grouping Option Target vs Sample Figure 28 Samples tab in Experiment Settings window To adjust the lists in these tabs use the following functions 39 Plates e Add atarget or sample name by typing a name in the New box and clicking Add e Remove a target or sample name from the list by clicking the Select to Remove box for that row and then clicking the Remove checked item s button e Select the target as a reference for gene expression data analysis by clicking the box in the Reference column next to the name for that target e Select the sample as a control sample for gene expression data analysis by clicking the box in the Control column next to the name for that sample Click the Show Analysis Settings box in the Experiment Settings window to view or change analysis parameters applied in the Gene Expression tab To adjust target parameters e Click a cell in the Color column to change the color of the targets graphed in the Gene Expression chart e Enter a number for the efficiency of a target The software will calculate the relative efficiency for a target using Auto Efficiency if the data for a target includes a standard curve Al
141. w Contents Show Symbols SR VO EER 3 4 5 6 7 8 3 m d n m 9 az 2 gt z z z a N i se I iiM Zi Zz zz e a om o fi o on oo n ehh a wilh w Figure 54 Trace Styles window Use the tools in the Trace Styles window to adjust appearance of traces and preview the changes in the well selector at the bottom of the window e Select a specific set of wells by using the well selector at the bottom of the window Alternatively select wells that contain one sample type in the pull down menu in the Wells column e Click the box in the Color column to select a color for the wells e Select a symbol from the pull down menu in the Symbol column e Click Show Contents to show the sample types in each well or click Show Symbols to show the selected Symbols in each well Log Scale Option Click the Log Scale box at the bottom of the Amplification chart to view the fluorescence traces in a semi log scale as shown in Figure 55 Amplifoaton FAM TET Figure 55 Log Scale option selected in Amplification chart 66 CFX96 and CFX96 Deep Well Systems Instruction Manual Standard Curve Chart The software creates a Standard Curve chart Figure 56 in the Quantitation tab if the data include sample types defined as standard Std for one fluorophore in the experiment Standard Curve Log Starting Quantity Stardard X Uko FAM E1238 R 2055 TET E 1000 R000
142. window to change the chart including changing the title selecting limits for the x and y axes showing grid lines and showing minor ticks in the axes NOTE Menu items that apply to specific charts are described in the next chapter Data Analysis Windows page 65 Spreadsheets 62 The spreadsheets shown in Data Analysis include options for sorting and transferring the data Sort the columns by one of these methods e Click and drag a column to a new location in the selected table e Click the column header to sort the data in Ascending or Descending order To sort up to three columns of data in the Sort window follow these steps 1 Right click on the spreadsheet to open the menu and select Sort 2 Inthe Sort window select the first column title to sort Sort the data in Ascending or Descending order 3 Select more than one column title by selecting the title in the pull down menu Select Ascending or Descending to sort the column in that order 4 Click 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 CFX96 and CFX96 Deep Well Systems Instruction Manual Common Right Click Menu Items for Spreadsheets Right click any spreadsheet view to select the items shown in Table 19 Table 19 Right click menu items for spread
143. x and press the Enter key To delete a sample name select it in the menu press the Delete key on your keyboard and then press Enter To load replicate numbers selected wells must contain identical well contents If they do not the software disables this loading control Click the Load box to add a Replicate to the selected wells 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 You can load replicate groups with replicate numbers progressing from left to right Horizontal or progressing from top to bottom Vertical 37 Plates Table 14 Options for loading the plate and wells in the Plate Editor Option Load Concentration Bite lt b w stating aac ne Concentration MOLE Bh Replicates from 1 to l3 Dilution Factor 10 Increasing Decreasing TIETE EA lt All v Well Note knone gt J E Collection Hame F hone gt yt i Experiment Settings Clear Replicate Bi Clear wells Function Enter a concentration to the selected wells with standard sample type by editing or typing a number in the Concentration box To apply the concentration to one fluorophore in the well select a single fluorophore from the pull down menu lt All gt under the c
144. y cycle Each trace in the chart represents data from a single fluorophore in one well Data Analysis GE File Marcom pcrd File view Settings Tools Fil Q A h view edit Plate ea Well Group All Wells Standard Curve 50 FA E 120 4 R 2 0 984 Slope 2 913 Log Scale HE E 100 0 R 2 0 000 Slope 0 000 ac oO mi a 0 o r Completed Scan Mode All Channels Plate Type BR Clear Analysis Mode Baseline Subtracted Curve Fit Figure 46 Layout for the Quantitation tab in the 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 CFX system can acquire fluorescence data at multiple protocol steps the software maintains the data acquired at each step independently The software displays the Step Number selector below the Standard Curve chart on the Quantitation tab whenever a protocol contains more than one data collection step When you select a step the software applies that selection to all the data that are shown in the Data Analysis window Figure 47 shows the data collection step number is 3 for all the data Step Number Figure 47 Step Number selection in the Data Analysis window o Data Analysis Overview Viewing Well Groups in Data Analysis Wells in

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