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Applied Biosystems 7500/7500 Fast Real

1. Notes 64 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment This chapter covers E LD OR so kg oa dida dados CASAS PI dd rikr A dA DS Ad DA 66 echo Review RO querer erre e450 edi a a dO SE DAS 67 E Analyze the Experiment icc csc deo 94S PS ds HOR ad a ES 68 E View the Standard Ce og ono kh oo 84S 45 6044 RI ES dd dA AS ORES ES T1 E View the Amplification Plot o an 0000s 74 E ere nel a oe ok sk pd dada E DI DO RS A 81 Wan a O capas pes asd he oe EE eh a oe dd 83 Section 5 2 Troubleshoot If Needed c cccccccccccc cc cccs 85 E View the Analysis Settings i onc ok 4a eds dA DES SA TU e E wR RRES 86 E view ie OL SUMMA srs rss rets dei PRA SARA APS Add 88 M Omit Wells from the Analysis 0 00 00 c ccc ccc eens 90 E View the Multicomponent Plot 0 0 0 0 00 92 E View the Raw Data Plot rsss b hohe bok eo iyini idiote iir da dd 94 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help gt 7500 Software Help Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 65 Experiments ma Chapter 5 Analyze the Experiment Chapter Overview Chapter Overview The 7500 so
2. Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 15 Experiments Chapter 5 Analyze the Experiment View the Amplification Plot 5 View the threshold values a In the Graph Type drop down list select Log b In the Target drop down list select RNase P TAMRA c Select the Threshold check box to show the threshold d Verify that the threshold is set correctly In the example experiment the threshold is in the exponential phase 5a Amplification Plot Plot Settings Plot Type ARn vs Cycle Graph Type Log v Plot Color Well Save current settings as the default e PARE ic Amplification Plot l 0 1 0 01 E 0 001 0 0001 0 00001 0 000001 0 0000001 H o 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Cycle Legend Mia Ms Mic MD ME Mr Mic MH Options Bb Target RNase P TAMRA Threshold 7 Auto 0 605352 Auto Baseline Bc Show Threshold Baseline Start Well E Target amp Baseline End Well E Target 4 Notes 76 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment nm View the Amplification Plot 6 Locate any outliers a b In the Plot Type drop down list select Cy vs Well Look for outliers in the amplification plot In the exampl
3. Dilution Point Source Source Volume uL Diluent Volume uL 1 10 000 2 5 000 The standard concentration in stock entered is too low to prepare the standard dilution series Enter a number greater 4 1 250 Assay Type Inventoried Made to Order v Reaction Volume Per Well 25 uL Excess Reaction Volume Print Reaction Setup 5 Reaction Setup Help A Total Volume uL Standard Concentrati f Inthe Standard Dilution Series for RNase P TAMRA pane see page 34 a Click the Standard Concentration in Stock field then enter 20000 b Click the units field then enter copies per UL Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 33 Experiments mem Chapter 2 Design the Standard Curve Experiment Review the Reaction Setup c Review the calculated volumes for preparing the standard dilution series Dilution Source Source Volume Diluent Volume Total Volume Standard Concentration Point HL HL HL ng uL 1 10000 Stock 3 63 14 52 18 15 4000 0 2 5000 Dilution 1 9 08 9 08 18 15 2000 0 3 2500 Dilution 2 9 08 9 08 18 15 1000 0 4 1250 Dilution 3 9 08 9 08 18 15 500 0 5 625 Dilution 4 9 0
4. Notes Consumable Consumable A MicroAmp Optical 96 Well Reaction Plate E MicroAmp Optical 8 Tube Strip 0 2 mL B Microamp Splash Free Support Base F MicroAmp Optical Tube without Cap C MicroAmp Optical 8 Cap Strip 0 2 mL G MicroAmp Optical Adhesive Film D MicroAmp Reaction Tube with Cap 0 2 mL 4 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments 7500 Fast System Notes Chapter 1 Get Started Supported Consumables The 7500 Fast system supports the consumables listed below IMPORTANT Use only Fast consumables reaction plates tube strips and tubes with the 7500 Fast system even when performing an experiment with standard reagents Consumable Part Number e MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 0 1 mL e 4346906 e MicroAmp Optical Adhesive Film e 4311971 e MicroAmp Fast 8 Tube Strip 0 1 mL e 4358293 e MicroAmp Optical 8 Cap Strip e 4323032 e Microamp Splash Free Support Base e N8010531 e MicroAmp Adhesive Film Applicator e 4333183 e MicroAmp Cap Installing Tool Handle e 4330015 e MicroAmp Multi Removal Tool e 4313950 E C 7 scescscsesg gt B Consumable Microamp Splash Free Support Base MicroAmp Fast Optical 96 Well Reaction Plate 0 1 mL MicroAmp Fast 8 Tube Strip A B C Micr
5. e Flag Settings tab to Adjust the sensitivity so that more wells or fewer wells are flagged Change the flags that are applied by the 7500 software Advanced Settings tab to change baseline settings well by well For More For more information on the analysis settings open the 7500 Software Help by pressing Information F1 when the Analysis Settings dialog box is open Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 87 Experiments m n Chapter 5 Analyze the Experiment no View the QC Summary View the QC Summary About the Example Experiment View the QC Summary The QC Summary screen displays a list of the 7500 software flags and includes the flag frequency and location for the open experiment In the standard curve example experiment you review the QC Summary screen for any flags generated by the experiment data In the example experiment well B12 produced a Cy that deviates significantly from the associated technical replicates generating the OUTLIERRG flag and indicating that the well is a potential outlier 1 In the navigation pane select Analysis gt QC Summary Note If no data are displayed click Analyze 2 Review the Flag Summary In the example experiment there is 1 flagged well 3 Inthe Flag Details table look in the Frequency and Wells columns to determine which flag appears in the experiment In the example experime
6. Set up the run method Review the reaction setup Order materials for the experiment 1 2 3 4 5 Set up the standards 6 7 8 9 1 O Finish the Design Wizard WwW Prepare the Reactions Chapter 3 v Run the Experiment Chapter 4 v Analyze the Experiment Chapter 5 v End Experiment 18 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment EN Create a New Experiment Create a New Experiment Create a new experiment using the Design Wizard in the 7500 software Create an 1 Double click 7500 software or select Start All Programs gt Applied Experiment Biosystems gt 7500 Software gt lt software name gt where lt software name gt is the current version of the 7500 software 2 In the Home screen click Design Wizard to open the Design Wizard File Edit Instrument Analysis Tools New Experiment f Open MM Save BR Close Export PrintReport Run Analyze Design Wizard QuickStart Analyze Experiment 2 Advanced Setup Create Study Template fe Home Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 19 Experiments ain Chapter 2 Design the Standard Curve Experiment Define the Experiment Properties Define the Experiment Properties In the Experiment Properties screen enter iden
7. 20 0 0 00 ccc eee o2 E View the Raw Data Plot oo oo hk 6 6 95 6S cagada SER CE DESLEAL dO OS 94 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 85 Experiments e Chapter 5 Analyze the Experiment View the Analysis Settings View the Analysis Settings The Analysis Settings dialog box displays the analysis settings for the threshold cycle Cr flags and advanced options If the default analysis settings in the 7500 software are not suitable for your experiment you can change the settings in the Analysis Settings dialog box then reanalyze your experiment About the In the standard curve example experiment the default analysis settings are used without Example changes Experiment View the Analysis 1 In the navigation pane select Analysis Settings 2 Click Analysis Settings to open the Analysis Settings dialog box 3 In the example experiment the default analysis settings are specified in the e Cy Settings tab e Flag Settings tab e Advanced Settings tab o Analysis Settings for standard curve example experiment Flag Settings Advanced Settings T Review the default settings for analysis of targets in this experiment To edit the default settings click Edit Default Settings To use different settings for a target select the target from the table deselect Use Default Settings then change the settings that are displayed Default Cr Se
8. Experiments Chapter 5 Analyze the Experiment View the Multicomponent Plot 6 Observe the FAM dye signal In the example experiment the FAM dye signal increases throughout the PCR process which indicates normal amplification VMiaws VAZA Tahlia Multicomponent Plot lt View Plate Layout 2a Plot Settings gt Select Wells With l Select item v l Select Item 3 1 Plot Color Dye v Show in Wells SE View Legend Save current settings as the default a BZ Multicomponent Plot 4 500 000 4 000 000 3 500 000 2b 3 000 000 2 500 000 2 000 000 Fluorescence 1 500 000 o 2 4 6 8 10 12 14 16 18 0 RKB BD LE EEE Cycle Legend E ROX DE TAMRA EH Fam Wells E 6 Unknown Ej 15 Standard 5 3 Negative Control 72 Empty f Select the negative control wells one at a time and check for amplification In the example experiment there is no amplification in the negative control wells Multicomponent Plot Plot Settings Plot Color Dye v Save current settings as the default Multicomponent Plot 4 500 000 4 000 000 3 500 000 3 000 000 2 500 000 2 000 000 Fluorescence 1 500 000 1 000 000 T 500 010 o A DE S EE TD RE DEDO eee ST Ce Coe meee es eee a eee cee res ees CI RS ome o 2 4 6 8 0 12 14 6 BMH Re BH BD RK BK BHM RL Cycle Legend MB ROX TAM
9. b In the Select Wells With drop down lists select Target then RNase P TAMRA 2a f View Plate Layout View Well Table 2b Select Wells With Selectiltem v EEE 3 Inthe Amplification Plot screen a In the Plot Type drop down list select ARn vs Cycle default b In the Plot Color drop down list select Well default Notes 14 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment View the Amplification Plot OP c Click Show a legend for the plot Note This is a toggle button When the legend is displayed the button changes to Hide the plot legend 4 View the baseline values a In the Graph Type drop down list select Linear b Select the Baseline check box to show the start cycle and end cycle c Verify that the baseline is set correctly The end cycle should be set a few cycles before the cycle number where significant fluorescence 1s detected In the example experiment the baseline is set correctly 3a 4a 3b Plot Settings Plot Type ARn vs Cycle Y gt Graph Type Linear Plot Color Well Save current settings as the default 2 P a g I se Amplification Plot 1 eee a E E a R e eee o 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Options Target 4b
10. notification settings 59 O OFFSCALE flag 89 124 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments omit wells 90 online Help See Help system order materials 37 other fluorescent based reagents 10 OUTLIERRG flag 89 outliers See omit wells overvoltage category rating xx P physical hazard safety xx prepare experiment for more information 46 50 54 guidelines 46 48 50 53 reaction mix prepare 49 reaction plate 51 sample dilutions 45 standard dilution series 47 workflow 44 prepare for run 57 print reaction setup instructions 35 publish data 83 Q QC Summary screen 88 QuickStart 11 100 R radioactive waste handling xix ramp speed 22 23 Raw Data Plot screen 94 reaction mix calculated volumes 33 volumes 49 reaction plate layout 13 40 load 57 prepare 51 unload from the instrument 64 reaction plates 6 Reaction Setup screen 32 reagents other fluorescent based 10 SYBR Green 9 TaqMan 9 repetitive motion safety xxi replicate component of experiment 7 results interpreting 67 run experiment alerts 63 Index monitor 61 prepare for 57 start 61 workflow 56 Run Method library 31 Run Method screen 30 monitor during arun 63 S safety before operating the instrument xvi biological hazards xxi chemical xvii chemical waste xix conventions Xii electrical xx ergonomic xxi guidelines xviii Xix instrument operation xvi
11. 1 RNase P reaction mix 74 25 8 25 b Mix the reaction by gently pipetting up and down then cap the tube c Centrifuge the tube briefly to remove air bubbles d Add 25 uL of the negative control reaction to the appropriate wells in the reaction plate 2 For each replicate group prepare the standard reactions a To appropriately sized tubes add the volumes of reaction mix and standard listed below f Reaction Mix Standard Volume Tube Standard Reaction Reaction Mix Volume uL Standard uL 1 RNase P Std 1 RNase P reaction mix 74 25 RNase P Std 1 8 25 2 RNase P Std 2 RNase P reaction mix 74 25 RNase P Std 2 8 25 3 RNase P Std 3 RNase P reaction mix 74 25 RNase P Std 3 8 25 4 RNase P Std 4 RNase P reaction mix 74 25 RNase P Std 4 8 25 5 RNase P Std 5 RNase P reaction mix 74 25 RNase P Std 5 8 25 b Mix the reactions by gently pipetting up and down then cap the tubes c Centrifuge the tubes briefly to remove air bubbles d Add 25 uL of the standard reaction to the appropriate wells in the reaction plate Notes 52 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 3 Prepare the Reactions 2 Prepare the Reaction Plate 3 For each replicate group prepare the reactions for the unknowns a To appropriately sized tubes add the volumes of reaction mix and sample listed below i Reacti
12. Analyze the Experiment Chapter 5 Section 1 Review Results Analyze View the standard curve View the amplification plot View the results in a table Publish the data Section 2 Review Multiple Experiments Results 0O A OO N Create a study Define replicates Analyze View the gene expression data View the experiment data View the multicomponent data View multiple plots View the quality summary Publish the data Section 3 Troubleshoot If Needed 1 View the analysis settings adjust the baseline threshold o ON OO FW N 2 View the quality summary 3 Omit wells 4 View the multicomponent plot 5 View the raw data plot v End Experiment Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 15 Experiments EN Chapter 1 Get Started Example Experiment Workflow Notes 16 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment This chapter covers E CDE ee so 66 cha dA dadas CARAS SOLA AHA AA HER ERED 18 Cha New DCD 6 gc obs oben psi pol oes re pie side eR PEA 19 E Define the Experiment Properties 44 445 00409 es esos geen sven dwn queda es 20 E Define the Methods and Materials 0 0 0 eee oe SU E ME oo 5 he hs Bhs de hb chee oe hs E E 24 Ro ot a oe ee ha oy en bi
13. Experiments Glossary A dye that produces fluorescence Because the passive reference signal should be consistent across all wells it is used to normalize the reporter dye signal to account for non PCR related fluorescence fluctuations caused by minor well to well differences in concentrations or volume Normalization to the passive reference signal allows for high data precision An illustration of the grid of wells and assigned content in the reaction plate In the 7500 7500 Fast system the grid contains 8 rows and 12 columns In the 7500 7500 Fast system software you can use the plate layout as a selection tool to assign well contents to view well assignments and to view results The plate layout can be printed included in a report exported and saved as a slide for a presentation One standard in a standard curve The standard quantity for each point in the standard curve is calculated based on the starting quantity and serial factor In genotyping experiments a DNA sample with a known genotype homozygous or heterozygous In the 7500 7500 Fast system software the task for the SNP assay in wells that contain a sample with a known genotype Used in genotyping and presence absence experiments the part of the instrument run that occurs after amplification In genotyping experiments fluorescence data collected during the post PCR read are displayed in the allelic discrimination plot and used to make allele calls In presence absenc
14. In the Password field enter password Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 59 Experiments A Chapter 4 Run the Experiment Enable the Notification Settings Optional Notification Settings Select the events to generate notifications Instrument Error 4 C Run Started z Run Completed 5 s Enter e mail addresses for notifications scientist mycompany com supervisor mycompany com technician mycompany com Separate e mail addresses with commas For example jane_smith mydomain com awong bigmailhost com 6 9 Qutgoing Mail Server SMTP smtp mycompany com For example smtp mycompany com Server requires an encrypted connection Yes No fa Server requires authentication Yes O No 7b Server Authentication User Name Example User Cc Server Authentication Password Run Guidelines When you set up the 7500 7500 Fast system for automatic notification e Your system must be set up for network use Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR Systems Maintenance Guide e Select the events for which you want to receive e mail notifications Select Instrument Error To notify recipients by email of all errors encountered by the instrument during each run Run Started To notify recipients by email when the instrument starts a run Run Comp
15. Note You can create a new experiment using the Design Wizard see Chapter 2 or Advanced Setup see page 98 3 Select File Export 4 Select the Export Properties tab default then a Select Setup b Select One File in the drop down list c Enter a name then select a location for the export file d Select E txt in the File Type drop down list IMPORTANT You cannot export xml files 5 Optional Select the Customize Export tab then select the appropriate options 6 Click Start Export f When prompted click Close Export Tool Create an You can import plate setup data from an exported text file txt to complete the reaction Experiment with plate setup data for your experiment an Exported Text File IMPORTANT Be sure the exported text file you select contains only reaction plate setup data and that the experiment types match 1 Import the reaction plate setup data from an exported text file a Using a spreadsheet application such as Microsoft Excel software open an exported text file b Replace the parameters of the text file as needed When finished save the file as a tab delimited text file Notes 104 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Appendix A Alternate Experiment Workflows A Export Import Workflow c In the Home screen click Advanced Setup Note If you do not see the Advanced Setup icon cl
16. a Using a new pipette tip add 9 08 uL of dilution 1 to the RNase P Std 2 tube b Vortex Std 2 for 3 to 5 seconds then centrifuge the tube briefly 5 Prepare dilution 3 in the RNase P Std 3 tube a Using a new pipette tip add 9 08 uL of dilution 2 to the RNase P Std 3 tube b Vortex Std 3 for 3 to 5 seconds then centrifuge the tube briefly 6 Prepare dilution 4 in the RNase P Std 4 tube a Using a new pipette tip add 9 08 uL of dilution 3 to the RNase P Std 4 tube b Vortex Std 4 for 3 to 5 seconds then centrifuge the tube briefly f Prepare dilution 5 in the RNase P Std 5 tube a Using a new pipette tip add 9 08 uL of dilution 4 to the RNase P Std 5 tube b Vortex Std 5 for 3 to 5 seconds then centrifuge the tube briefly 8 Place the standards on ice until you prepare the reaction plate Preparation When you prepare your own standard curve experiment Guidelines e Standards are critical for accurate analysis of run data e Any mistakes or inaccuracies in making the dilutions directly affect the quality of results e The quality of pipettors and tips and the care used in measuring and mixing dilutions affect accuracy e Use TE buffer or water to dilute the standards Notes 48 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 3 Prepare the Reactions fate Prepare the Reaction Mix Prepare the Reaction Mix Prepare the reactio
17. gt 7500 Software Help in the 7500 software e Genotyping experiments Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Genotyping Experiments e Presence absence experiments Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Presence Absence Experiments e Relative standard curve and or comparative Cy AAC experiments Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR Systems Getting Started Guide for Relative Standard Curve and Comparative C Experiments Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 3 Experiments re Chapter 1 Get Started Supported Consumables Supported Consumables 7500 System The 7500 system supports the consumables listed below Consumable Part Number e MicroAmp Optical 96 Well Reaction Plate with Barcode 0 2 mL e 4306737 e MicroAmp Optical Adhesive Film e 4311971 e MicroAmp Optical 8 Tube Strip 0 2 mL e 4316567 e MicroAmp Optical 8 Cap Strip e 4323032 e MicroAmp Optical Tube without Cap 0 2 mL e N8010933 e MicroAmp Reaction Tube with Cap 0 2 mL e N8010540 e Microamp Splash Free Support Base e N8010531 e MicroAmp Adhesive Film Applicator e 4333183 e MicroAmp Cap Installing Tool Handle e 4330015 e MicroAmp Multi Removal Tool e 4313950 D B C F B
18. 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 1 Get Started santos How to Use This Guide How to Use This Guide This guide functions as both a tutorial and as a guide for performing your own experiments Using This Guide By using the example experiment data provided with the 7500 software you can use this as a Tutorial guide as a tutorial for performing a standard curve experiment on a 7500 7500 Fast system Follow the procedures in Chapters 2 through 5 Chapter Procedure 2 Design the experiment using the Design Wizard in the 7500 software 3 Prepare the experiment using the reagents and volumes calculated by the Design Wizard in Chapter 2 4 Run the experiment on a 7500 7500 Fast instrument 5 Analyze the results For more information see About the Example Standard Curve Experiment on page 12 Using This Guide After completing the tutorial exercises in Chapters 2 through 5 you can use this guide to with Your Own lead you through your own standard curve experiments Each procedure in Chapters 2 Experiments through 5 includes a set of guidelines that you can use to perform your own experiments Additionally you can use one of the other workflows provided in the 7500 software to perform your experiments The table below provides a summary of all the workflows available in the 7500 software Workflow Description See Design Wizard Set u
19. ANITY CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument AN WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDsSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain MSDSs 1 Go to www appliedbiosystems com click Support then click MSDS Search 2 In the Keyword
20. Biosystems to TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assays Data file on a CD shipped with each assay order The file name includes the number from the barcode on the plate The information in the AIF is provided in a tab delimited format PCR reaction component in Applied Biosystems TaqMan Gene Expression Assays and TagMan SNP Genotyping Assays The assay mix contains primers designed to amplify a target and a TaqMan probe designed to detect amplification of the target In the run method a setting to increase or decrease the temperature and or time for a step with each subsequent cycle in a cycling stage When AutoDelta is enabled for a cycling stage the settings are indicated by an icon in the thermal profile e AutoDelta on A e AutoDelta off A An analysis setting in which the software calculates the baseline start and end values for the amplification plot You can apply the automatic baseline setting to specific wells in the reaction plate See also baseline An analysis setting in which the software calculates the baseline start and end values and the threshold in the amplification plot The software uses the baseline and threshold to calculate the threshold cycle Cy See also threshold cycle CT In the amplification plot a line fit to the fluorescence levels during the initial stages of PCR when there is little change in fluorescence Applied Biosystems 7500 7500 Fast Real Time PCR System Getting
21. Chapter 5 Analyze the Experiment View the Multicomponent Plot View the Multicomponent Plot About the Example Experiment View the Multicomponent Plot Notes The Multicomponent Plot screen displays the complete spectral contribution of each dye in a selected well over the duration of the PCR run In the standard curve example experiment you review the Multicomponent Plot screen for In the navigation pane select Analysis gt ROX dye passive reference FAM dye reporter Spikes dips and or sudden changes Amplification in the negative control wells Multicomponent Plot Note If no data are displayed click Analyze Display the unknown and standard wells one at a time in the Multicomponent Plot screen a Select the View Plate Layout tab b Select one well in the plate layout the well is shown in the Multicomponent Plot screen Note If you select multiple wells the Multicomponent Plot screen displays the data for all selected wells simultaneously In the Plot Color drop down list select Dye Click 1 Show a legend for the plot Note This is a toggle button When the legend is displayed the button changes to Hide the plot legend Observe the ROX dye signal In the example experiment the ROX dye signal remains constant throughout the PCR process which indicates typical data 92 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve
22. Defaults tab default In the Default Instrument Type drop down list select the appropriate instrument Preferences Defaults Startup Enter a default sample volume for your experiments select your default folders and select the type of instrument for the software to use by default Sample Reaction Vol 50 uL Data Folder C Applied Biosystems 7500 experiments Browse Import Folder C Applied Biosystems 7 500 experiments Browse Export Folder C AApplied Biosystems 7500 experiments Browse Instrument Type 7500 v OK Cancel For More For more information on Information Notes Completing the Experiment Properties screen Open the 7500 Software Help by clicking or pressing F1 Consumables See Supported Consumables on page 4 Quantitation experiments Refer to the Applied Biosystems Real Time PCR Systems Reagent Guide Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 21 Experiments aie Chapter 2 Design the Standard Curve Experiment L Define the Methods and Materials Define the Methods and Materials In the Methods amp Materials screen select the quantitation method reagents ramp speed and PCR template to use for the experiment About the In the standard curve example experiment Example r h The standard curve quantitation method is used Experiment TaqMan reagents are used e The sta
23. Experiment Set Up the Samples Set Up the Samples About the Example Experiment Complete the Samples Screen Notes In the Samples screen enter the number of samples replicates and negative controls to include in the reaction plate enter the sample names then select the sample target reactions to set up In the standard curve example experiment Two samples are used genomic DNA from two populations The samples contain unknown quantities of the target RNase P Three replicates are used The replicates are identical reactions containing identical reaction components and volumes Three negative controls are used The negative control reactions contain water instead of sample and should not amplify Click the How many samples do you want to test in the reaction plate field then enter 2 Note The number of rows in the samples table is updated with the number you entered Click the How many replicates do you need field then enter 3 Click the How many negative controls do you need for each target assay field then enter 3 Set up Sample 1 a Click the Enter Sample Name field then enter pop1 for population 1 b In the Color field leave the default Set up Sample 2 a Click the Enter Sample Name field then enter pop2 for population 2 b In the Color field leave the default Select No Biological Replicates default Select All Sample Target Reactions to test all targets in all samples de
24. Mean 28 260696 28 260696 28 260696 27 200424 27 200424 27 200424 26 281769 26 281769 26 281769 27 277845 27 277845 27 277845 28 434366 28 434366 28 434366 29 538963 29 538963 29 538963 30 697205 30 697205 30 697205 Cr SD 0 02 0 02 0 02 0 036 0 036 0 036 0 094 0 094 0 094 0 082 0 082 0 082 0 228 0 228 0 228 0 114 0 114 0 114 0 247 0 247 0 247 Quantity 2 772 365 2 841 162 2 808 998 5 308 388 5 496 287 5 535 287 10 000 10 000 10 000 5 000 5 000 5 000 2 500 2 500 2 500 1 250 1 250 1 250 625 625 625 Quantity 2 807 509 2 807 509 2 807 509 5 446 654 5 446 654 5 446 654 Quantity 34 422 34 422 34 422 121 319 121 319 121 319 A 82 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment no Publish the Data Analysis When you analyze your own standard curve experiment group the wells by Guidelines e Replicate The software groups the wells by replicate negative controls standards and samples Look in the Quantity columns to make sure the quantity values for each replicate group are similar This indicates tight precision e Flag The software groups the flagged and unflagged wells A flag indicates that the software found an error in the flagged well For a description of the 7500 software flags see View the QC Summary on page 88 e C
25. Mix dilution factor dissociation curve EFF endogenous control endpoint read experiment 112 A process during the instrument run in which an instrument component detects fluorescence data from each well of the reaction plate The instrument transforms the signal to electronic data and the data are saved in the experiment file In the 7500 7500 Fast system software a data collection point is indicated by an icon in the thermal profile e Data collection on e Data collection off See baseline corrected normalized reporter DRn The negative first derivative of the normalized fluorescence generated by the reporter during PCR amplification In the derivative reporter Rn vs temperature melt curve the derivative reporter signal is displayed in the y axis A feature in the 7500 7500 Fast system software that helps you set up your experiment by guiding you through best practices as you enter your experiment design A reagent used to dilute a sample or standard before adding it to the PCR reaction The diluent can be water or buffer In the 7500 7500 Fast system software a field displayed on the Sample Dilution Calculations tab of the Reaction Setup screen For this field enter the sample concentration you want to use to add to the reaction mix for all samples in the experiment 10X for Reaction Mix indicates that the software assumes the sample or standard component of the reaction mix is at a 10X concentration Fo
26. Mix Concentration field displays 20 0X default 32 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment Review the Reaction Setup aoe c Review the components and calculated volumes for the PCR reactions Component Volume for 1 Reaction pL Master Mix 2 0X 12 50 Assay Mix 20 0 1 25 Sample 10X or Standard 2 50 H O 8 75 Total Volume 25 00 The sample or standard volume is limited to 10 of the total reaction volume 3 2E Set Up Reaction Setup For each target assay in the reaction plate selej tthe assay type if using TaqMan reagents then review the calculated volumes for preparing the standard dilution series T Instructions samples and PCR reactions If needed edit thi reaction volume excess reaction volume component concentrations and or stock concentrations Click Print Reaction Setup to print instructions on how to prepare thj2 PCR reactions 1 Reaction Mix Calculations Sample Dilutior Calculations Select Target RNase P TAMRA Reactions for RNase P TAMRA Master Mix Concentration X Assay Mix Concentration 20 0 X Volume uL for 1 Reaction Component 6c Standard Dilution Series for RNase P TAMRA Standard Concentration in Stock 100 0 ng w peruLl
27. Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following e Open To view the document e Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve xvii Experiments Preface Chemical Safety Chemical Safety To minimize the hazards of chemicals xviii Guidelines Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page xvii Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fame hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or
28. Select the Set Up Standards check box Applied Biosystems recommends that you set up a standard curve for each target experiment in the reaction plate e Identify each target experiment with a unique name and color You can enter up to 100 characters in the Target Name field e Select the reporter dye used in the target experiment In the Methods amp Materials screen on page 22 if you selected TaqMan Reagents Select the dye attached to the 5 end of the TaqMan probe SYBR Green Reagents Select SYBR e Select the quencher used in the target experiment In the Methods amp Materials screen on page 22 if you selected TaqMan Reagents Select the quencher attached to the 3 end of the TaqMan probe SYBR Green Reagents Select None For More For more information on completing the Targets screen open the 7500 Software Help by Information clicking or pressing F1 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 25 Experiments ni Chapter 2 Design the Standard Curve Experiment Set Up the Standards Set Up the Standards In the Standards screen enter the number of points and replicates for all standard curves in the reaction plate For each standard curve enter the starting quantity and select the serial factor About the In the standard curve example experiment Example Experiment Complete the 1 Standards Screen 3b Notes One
29. Started Guide for Standard Curve Experiments baseline corrected normalized reporter ARn biological replicates blocked IPC Cr calibrator chemistry comparative C AAC method custom dye cycle threshold cycling stage Glossary The magnitude of normalized fluorescence generated by the reporter 1 In experiments that contain data from real time PCR the magnitude of normalized fluorescence generated by the reporter at each cycle during the PCR amplification In the ARn vs Cycle amplification plot ARn is calculated at each cycle as ARn cycle Rn cycle Rn baseline where Rn normalized reporter 2 In genotyping experiments and presence absence experiments the difference in normalized fluorescence generated by the reporter between the pre PCR read and the post PCR read In the allelic discrimination plot genotyping experiments and the presence absence plot presence absence experiments ARn is calculated as ARn Rn post PCR read Rn pre PCR read where Rn normalized reporter See also normalized reporter Rn Reactions that contain identical components and volumes but evaluate separate samples of the same biological source for example samples from three different mice of the same strain or separate extractions of the same cell line or tissue sample See also technical replicates In presence absence experiments a reaction that contains IPC blocking agent which blocks amplif
30. Started Guide for Standard Curve 49 Experiments NQ Chapter 3 Prepare the Reactions Prepare the Reaction Mix AN Maio CHEMICAL HAZARD TaqMan 2X Universal PCR Master Mix No AmpErase UNG may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 1 Label an appropriately sized tube for the reaction mix RNase P Reaction Mix 2 For the RNase P assay add the required volumes of each component to the tube Gamoanent Volume for Volume for 24 Reactions Plus p 1 Reaction uL 10 Excess uL TaqMan Universal PCR Master Mix 2X or 12 50 330 0 TaqMan 2X Universal PCR Master Mix No AmpErase UNG RNase P Assay Mix 20X 1 25 33 0 Water 8 75 231 0 Total Reaction Mix Volume 22 50 594 0 3 Mix the reaction mix by gently pipetting up and down then cap the tube 4 Centrifuge the tube briefly to remove air bubbles 5 Place the reaction mix on ice until you prepare the reaction plate Preparation When you prepare your own standard curve experiment Guidelines e If your experiment includes more than one target assay prepare the reaction mix for each target assay separately e Include excess volume in your calculations to provide excess volume for the loss that occurs during reagent transfers Applied Biosystems recommends an excess volume of at least 10
31. The threshold cycle C is the PCR cycle number at which the fluorescence level meets the threshold A C value gt 8 and lt 35 is desirable A C value lt 8 indicates that there is too much template in the reaction A Cy value gt 35 indicates a low amount of target in the reaction for Cy values gt 35 expect a higher standard deviation For More For more information on the Well Table open the 7500 Software Help by clicking or Information pressing F1 Publish the Data You can publish the experiment data in several ways e Save the plot as an image file e Print the plot e Print the plate layout e Create slides e Print a report e Export data For information on performing these procedures open the 7500 Software Help by clicking or pressing F1 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 83 Experiments Chapter 5 Analyze the Experiment a Publish the Data Notes 84 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment Section 5 2 Troubleshoot If Needed Section 5 2 Troubleshoot If Needed This section covers E View the Analysis Settings i one 0k 6 eee es a SEWERS RE SATA ROOTES RSE RES 86 E View the OC a oe hh 4 he ESS Ee OK EE H DA GER da 88 E Omit Wells from the Analysis 0 00 00 ccc eens 90 E View the Multicomponent Plot
32. Time PCR System Getting Started Guide for Standard Curve 43 Experiments a Chapter 3 Prepare the Reactions Chapter Overview Chapter Overview This chapter explains how to prepare the PCR reactions for the standard curve example experiment and provides guidelines for how to prepare the PCR reactions for your own standard curve experiment Example Start Experiment Experiment v Workflow Design the Experiment Chapter 2 wW Prepare the Reactions Chapter 3 Prepare the sample dilutions Prepare the standard dilution series Prepare the reaction mix for each target assay KR WwW N Prepare the reaction plate v Run the Experiment Chapter 4 v Analyze the Experiment Chapter 5 End Experiment Notes 44 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 3 Prepare the Reactions iain Prepare the Sample Dilutions Prepare the Sample Dilutions Perform sample dilutions before adding the samples to the final reaction mix Dilute the samples using the volumes that were calculated by the 7500 software Complete the Sample Dilution Calculations Tab on page 34 About the For the standard curve example experiment Example i Sample dilutions are necessary because the sample volume is limited to 10 of the Experiment total reaction volume in the 7500 software Because the total reacti
33. connection Product availability and pricing may vary according to your region or country Online ordering through the Applied Biosystems Store is not available in all countries Contact your local Applied Biosystems representative for help Note The 7500 software recommends the materials to order based on your experiment design It is assumed that you will design your experiment order your materials then prepare Chapter 3 and run Chapter 4 the reaction plate when your materials arrive About the In the standard curve example experiment the recommended materials are Example e MicroAmp Optical 96 Well Reaction Plate exponen e MicroAmp Optical Adhesive Film e MicroAmp Splash Free Support Base e TaqMan Universal PCR Master Mix 2X or TaqMan Universal PCR Master Mix No Amperase UNG e Applied Biosystems RNase P assay Complete the 1 For the example experiment leave the Find Assay pane empty Materials List You can order the human RNase P FAM dye labeled probe from Applied Screen Biosystems as the TaqMan RNase P Detection Reagents FAM dye PN 4316831 Note When you design your own standard curve experiment see Design Guidelines on page 39 for information on how to complete the Find Assay pane 2 Inthe Display drop down list select All Items default then review the recommended materials If needed use the scroll bar at right to see all items Note For more information on a specific item click
34. create Using the Design Wizard another experiment using the Design Wizard Return to the Wizard Return to the experiment to make changes using the Design Wizard e By default experiments are saved to lt drive gt Applied Biosystems lt software name gt experiments To change the Save location for a specific experiment Navigate to the desired location using the Save Experiment dialog box Default save location Select Tools Preferences then select the General tab In the Data Folder field browse to then select the desired location For More For more information on using Advanced Setup see Advanced Setup Workflow on Information page 98 Notes 42 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Notes Chapter 3 Prepare the Reactions This chapter covers DN Ol aa ee nr pad Ea ae ee eee A ee ee 44 E Prepare ihe Sample Des sans psd ewes dew eens d PE CS Li 45 E Prepare the Standard Dilution Series 0 0 ccc ees 47 E Prepare the Reaction Mess pera sda 6 Rd Sd dA ONE RE 49 CGC DC ROC TRE carris de paira PE Ed RUM A gd 51 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help gt 7500 Software Help Applied Biosystems 7500 7500 Fast Real
35. dd 64 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500 Software Help Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 95 Experiments A Chapter 4 Run the Experiment Chapter Overview Chapter Overview This chapter explains how to perform a run on the Applied Biosystems 7500 7500 Fast Real Time PCR System Example Start Experiment Experiment v Workflow Design the Experiment Chapter 2 v Prepare the Experiment Chapter 3 v Run the Experiment Chapter 4 Prepare for the run Optional Enable the notification settings Start the run Monitor the run O A OO N lt a Unload the instrument v Analyze the Experiment Chapter 5 D End Experiment Notes 56 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 4 Run the Experiment A Prepare for the Run Prepare for the Run Prepare for the run by opening the example experiment file you created in Chapter 2 then loading the sealed reaction plate into the 7500 7500 Fast instrument Open the 1 Double click 3 7500 software or select Start All Programs Applied Example Biosystems gt 7500 Software gt lt softwar
36. is switched receive or supply alternating on to the Standby condition current or voltage Hazardous voltage may be present if this switch is on standby Indicates the On Off position of a TEND Indicates a terminal that can push push main power switch FRED receive or supply alternating or direct current or voltage Safety Symbols The following table describes the safety symbols that may be displayed on Applied Biosystems instruments Each symbol may appear by itself or in combination with text that explains the relevant hazard see Safety Labels on Instruments on page xiv These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Description appropriate caution Indicates that you should consult the manual for further information and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution appropriate caution Indicates the presence of a laser inside the instrument and to proceed with rere gt gt I caution Indicates the presence of moving parts and to proceed with appropriate Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments xiii Preface Safety Labels on Instruments Enviro
37. limitations may apply Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve xix Experiments Preface Flectrical Safety Electrical Safety Fuses Power Overvoltage Rating pyleiaggd ELECTRICAL SHOCK HAZARD Severe electrical shock can result from operating the Applied Biosystems 7500 7500 Fast Real Time PCR System without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument LAN FIRE HAZARD Improper fuses or high voltage supply can damage the instrument wiring system and cause a fire Before turning on the instrument verify that the fuses are properly installed and that the instrument voltage matches the power supply in your laboratory WEGA FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument DANa ELECTRICAL HAZARD Grounding circuit continuity is vital for the safe operation of equipment Never operate equipment with the grounding conductor disconnected DANNE ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage supply in your facility aleis ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity The Applied Biosystems 7500 7500 Fast Real Time PCR System has an installation
38. of the 7500 software Open an existing experiment or create a new experiment Note You can create a new experiment using the Design Wizard see Chapter 2 or Advanced Setup see page 98 Select File Save As Template Enter a file name select a location for the template then click Save Click Close In the Home screen click Template Note If you do not see the Template icon click the arrow beneath the Design Wizard icon to expand the Set Up menu Locate then select the template you created in steps 1 through 5 above then click Open A new experiment is created using the following setup information from the template e Experiment properties e Plate setup e Run method Reaction setup Optional If you want to modify the experiment use Advanced Setup see page 98 Click Save enter a file name then click Save to save the experiment 102 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments o Prepare the PCR reactions Appendix A Alternate Experiment Workflows A Experiment Type Prepare the Relative standard curve Standard curve Template Sample dilutions Template Workflow Standard dilution series Reaction mix Reaction plate o OOo Too Comparative Cr a Template Genotyping b Sampie Cuong c Reaction mix Presence absence d Reaction plate 6 Run the experime
39. other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS Applera Applied Biosystems AB Design MicroAmp Primer Express and VIC are registered trademarks and FAM JOE ROX and TAMRA are trademarks of Applied Biosystems or its subsidiaries in the U S and or certain other countries AmpErase AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc SYBR is a registered trademark of Molecular Probes Inc Macintosh is a registered trademark of Apple Computer Inc Microsoft and Windows are registered trademarks of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4387779 Rev C 06 2010 Contents PICIACO quartas E DS toon de SE E a eae eens vil How to Use This Guide 0 ccc eee eee eee vii How to Obtain More Information 0 00 e Ix How to Obtain SUDDOM suga anna ide ew eb eGo edo ded AD ee Reese aw xi Safety Conventions Used in This Document 0000 cee eee eee ees xii Symbols on Instruments 000 eee ee xili Safety Labels on Instruments cccccccccccc eee eee XIV General Instrument Safety 0 0 eee eee xvi Chemical Salely yers orense EEIE hash eer NEEESE E
40. overvoltage category of II and is classified as portable equipment Physical Hazard Safety Moving Parts XX AD eee PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface Biological Hazard Safety Biological Hazard Safety General AN WARNING BIOHAZARD Biological samples such as tissues body fluids Biohazard infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occup
41. p30 RNaseP protein p30 RNas Gene rCG48258 Celera Annotation similar to Ribonuclease P protein subunit p30 RNaseP protein p30 RNas Gene rCG48258 Celera Annotation RefSeq NM_080010 2 NM_080010 2 NM_080010 2 NM_080010 2 XM_001063377 1 XM_001063377 1 XM_001063377 1 Next gt Last Cancel For More For more information on completing the Materials List screen open the 7500 Software Help by clicking or pressing F1 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments mam Chapter 2 Design the Standard Curve Experiment L Finish the Design Wizard Finish the Design Wizard To finish the Design Wizard review the plate layout then select an exit option About the The 7500 software automatically selects locations for the wells in the reaction plate In Example the standard curve example experiment Experiment e The wells are arranged as shown below po Rai Rai po pi PaE H RNase S RNase g RNase A N RNase N RNase N RNase m gase m Rae m Riuse m RNase D RNase v RNase 27763 o oies AC SIES Asses Asses Assacs AE 1E4 1E4 g RNase H RNase g RNase g RNase 5 RNase 5 RNase 5 RNase 5 RNase S RNase g RNase S RNase g RNase 5E3 SER SER 2 563 2 563 2 563 1 2563 1 2563 1 2563 625 625 625 Wells E 6 Unknown E 15 Standard 9 3 Negative Control 72 Empty e The experiment is save
42. pipetting inaccuracies include replicates e PCR inhibitors PCR inhibitors in the reaction can reduce amplification and alter measurements of the efficiency Display of data collected during the cycling stage of PCR amplification Can be viewed as e Baseline corrected normalized reporter ARn vs cycle e Normalized reporter Rn vs cycle e Threshold cycle C vs well Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 109 Experiments Glossary amplification stage assay Assay ID assay information file AIF assay mix AutoDelta automatic baseline automatic Cr baseline 110 Part of the instrument run in which PCR produces amplification of the target The amplification stage is called a cycling stage in the thermal profile and consists of denaturing primer annealing and polymerization steps that are repeated For quantitation experiments fluorescence data collected during the amplification stage are displayed in an amplification plot and the data are used to calculate results For genotyping or presence absence experiments fluorescence data collected during the amplification stage are displayed in an amplification plot and the data can be used for troubleshooting See also cycling stage In the 7500 7500 Fast system a PCR reaction mix that contains primers to amplify a target and a reagent to detect the amplified target Identifier assigned by Applied
43. provides you with maximum flexibility in the design and setup of your experiment See assay information file AIF For a given target any of the different sequences that occurs in the population Display of data collected during the post PCR read The allelic discrimination plot is a graph of the normalized reporter signal from the allele 1 probe plotted against the normalized reporter signal from the allele 2 probe A segment of DNA amplified during PCR Part of the instrument run in which PCR produces amplification of the target For quantitation experiments fluorescence data collected during amplification are displayed in an amplification plot and the data are used to calculate results For genotyping or presence absence experiments fluorescence data collected during amplification are displayed in an amplification plot and the data can be used for troubleshooting Calculation of efficiency of the PCR amplification The amplification efficiency is calculated using the slope of the regression line in the standard curve A slope close to 3 32 indicates optimal 100 PCR amplification efficiency Factors that affect amplification efficiency e Range of standard quantities To increase the accuracy and precision of the efficiency measurement use a broad range of standard quantities 5 to 6 logs 10 to 10 fold Number of standard replicates To increase the precision of the standard quantities and decrease the effects of
44. to determine the quantity of the RNase P gene in two populations In the standard curve example experiment The samples are genomic DNA isolated from two populations The target is the RNase P gene One standard curve is set up for the RNase P gene target The standard used for the standard dilution series contains known quantities of the RNase P gene Because a single target is being studied only one standard curve is required Note In experiments where multiple targets are being studied a standard curve is required for each target Three replicates of each sample and each dilution point in the standard curve are performed to ensure statistical significance The experiment is designed for singleplex PCR where every well contains a primer probe set for a single target Primer probe sets are from Applied Biosystems RNase P assay Note The human RNase P FAM dye labeled MGB probe is not available as a TaqMan Gene Expression Assay It can be ordered as a Custom TaqMan Gene Expression Assay PN 4331348 12 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 1 Get Started About the Example Standard Curve Experiment Reaction Plate The 7500 software displays the 96 well reaction plate layout as shown below Layout pop pop pop pop pop pop RN RN RH A R RNase R RNase R RNase m ggas m a m oe m RNase m RNase m Rilase El B El a E
45. user documentation For more information see Safety Words Alert Words on page xii How to Obtain More Information Related Documents Related to Genotyping Experiments Documentation Document PN Allelic Discrimination Pre Developed TaqMan Assay Reagents Quick Reference 4312212 Card Custom TaqMan Genomic Assays Protocol Submission Guidelines 4367671 Custom TaqMan SNP Genotyping Assays Protocol 4334431 Ordering TagqMan SNP Genotyping Assays Quick Reference Card 4374204 Performing a Custom TaqMan SNP Genotyping Assay for 96 Well Plates Quick 4371394 Reference Card Performing a TaqMan Drug Metabolism Genotyping Assay for 96 Well Plates 4367636 Quick Reference Card Pre Developed TaqMan Assay Reagents Allelic Discrimination Protocol 4312214 TaqMan Drug Metabolism Genotyping Assays Protocol 4362038 TaqMan SNP Genotyping Assays Protocol 4332856 Documents Related to Presence Absence Experiments Document PN DNA Isolation from Fresh and Frozen Blood Tissue Culture Cells and Buccal 4343586 Swabs Protocol Nicaea Chemistry Isolation of Genomic DNA from Animal and Plant Tissue 4333959 rotoco Documents Related to Relative Standard Curve and Comparative C Experiments Document PN Amplification Efficiency of TagMan Gene Expression Assays Application Note 127AP05 Applied Biosystems High Capacity cDNA Reverse Transcription Kits Protocol 4375575 Custom TaqM
46. with the Applied Biosystems Store click Register Now to create an account Order Materials Log In Log into the Applied Biosystems Store to place the selected items in your shopping basket If you do not have a user name and password click Register Now to create a new account OR i Store Log In Register To log into the Applied Biosystems Store enter your user Ifyou do not have name and password then click Log In and Submit an Applied 22 Biosystems User Name account click the i link below to create a new 5b Password account Log In and Submit Register Now C Remember my user name and password for future orders c When you are connected to the Applied Biosystems Store follow the prompts to complete your order 6 Go to Finish the Design Wizard on page 40 Notes 38 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment o Order Materials for the Experiment Design When you design your own standard curve experiment idelin i Guidelines Select all the materials that you require for your experiment then add them to your shopping list e To access and use the Applied Biosystems Store Confirm that your computer has an Internet connection Use the following Applied Biosystems recommended browsers and versions of Adobe Acro
47. 00 Fast Real Time PCR System Getting Started Guide for Standard Curve 89 Experiments Chapter 5 Analyze the Experiment Omit Wells from the Analysis Omit Wells from the Analysis Experimental error may cause some wells to be amplified insufficiently or not at all These wells typically produce C values that differ significantly from the average for the associated replicate wells If included in the calculations these outliers can result in erroneous measurements To ensure precision omit the outliers from the analysis About the In the standard curve example experiment well B12 is flagged as a potential outlier Example Experiment Omit Wells 1 In the navigation pane select Analysis gt Amplification Plot Note If no data are displayed click Analyze 2 Inthe Amplification Plot screen select Cy vs Well in the Plot Type drop down list 3 Select the View Well Table tab 4 Inthe Well Table a In the Group By drop down list select Replicate b Look for any outliers in the replicate group be sure they are flagged In the example experiment the 7500 software flagged well B12 as a potential outlier 3 View Plate Layout View Ei Table 4a Select Wells With Selectitem Well Omit Flag Sample Target N Task Dyes GT CT Mean CTSD Quantity Quantity Quantity RNase P TAMRA NTC a 1 Al d RNase P T NTC FAM TAMRA Undetermi 2 A2 RNase PT NTC FAM TAMRA Und
48. 00 software you can set up the experiment according to your own design 1 Double click 3 7500 software or select Start gt All Programs gt Applied Biosystems gt 7500 Software gt lt software name gt where lt software name gt is the current version of the 7500 software 2 Inthe Home screen click Advanced Setup Note If you do not see the Advanced Setup icon click the arrow beneath the Design Wizard icon to expand the Set Up menu 3 To set up a new experiment a Click Experiment Properties default enter the experiment name then select the experiment properties b Click Plate Setup Experiment Type Action Genotyping Define the SNP assays then assign them to wells in the reaction plate All other experiments Define the targets then assign them to wells in the reaction plate c Click Add Biological Group to create biological replicates and assign replicates to samples for each biological group in the reaction plate d Click Run Method review the reaction volume and thermal profile then edit as needed e Click 4 Reaction Setup review the components and calculated volumes for the PCR reactions then edit as needed f Optional Click Materials List review the list of materials then order the materials you need to prepare the reaction plate Notes 98 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve E
49. 500 software Comparative Ct Example eds Comparative Ct Study Example edm Comparative Ct Study Biological Groups edm Genotyping Example eds Presence Absence Example eds Relative Standard Curve Example eds Standard Curve Example eds IMPORTANT Be sure to use the Standard Curve Example eds file when you perform the procedures in this guide Example Experiment Workflow Notes Start Experiment v OA NOOK WODND O Design the Experiment Chapter 2 Create a new experiment Define the experiment properties Define the methods and materials Set up the targets Set up the standards Set up the samples Set up the run method Review the reaction setup Order materials for the experiment Finish the Design Wizard v AA OO N Prepare the Reactions Chapter 3 Prepare the sample dilutions Prepare the standard dilution series Prepare the reaction mix for each target assay Prepare the reaction plate v see page 15 14 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 1 Get Started Ao Example Experiment Workflow see page 14 WwW Run the Experiment Chapter 4 Prepare for the run Enable the notification settings Optional Start the run Monitor the run O A Ww N Unload the instrument and transfer the data v
50. 695 ic WO s WG W a E E E T ae can ee Sater TR Cycle Threshold Set Too High GE The threshold is set above the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Drag the threshold bar down into the exponential phase of the curve 0 14 Task Dyes Er CrMgan CrSD Quantity Quanity Quantity Comme E NTE FAM NEO Undesemil qr HTE FAM HEG Undedemi NTE FAM NFO Undedermi pary UNKNOWN FAMENFO 37681107 3745507 0202 2571 177 2888425 353775 l UMENGAH FAM HFG 37 27971 3749407 0 230 3260623 2866425 383 175 UMENG FAM MEG 37 524355 3749507 0 202 2824187 27809 42 353 775 0 001 0 0004 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 19 Experiments ane Chapter 5 Analyze the Experiment View the Amplification Plot Baseline Examples Baseline Set Correctly The amplification curve begins after the maximum baseline Baseline Set Too Low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value Baseline Set Too High The amplification curve begins before the maximum baseline Decrease the End Cycle value If your experiment does not meet the guidelines above e Omit wells see Omit Wells from the Analysis on page 90 or Manually adjust the baseline and or threshol
51. 8 9 08 18 15 250 0 7a Standard Dilution Series for RNase P TAMRA Standard Concentration in Stock 0000 0 copies peryL Dilution Point Source Source Volume uL Diluent Volume uL Total Volume uL Standard Concentration ng uL 1 10 000 Stock 3 63 14 52 18 15 4000 0 Complete the Sample Dilution Calculations Tab 1 Select the Sample Dilution Calculations tab 2 Click the Diluted Sample Concentration 10X for Reaction Mix field then enter 6 6 3 Inthe unit drop down list select ng uL default 4 Review the calculated volumes for the sample dilutions Sample Stock Concentration Sample Volume Diluent Volume Total Volume of Diluted Sample Name ng uL HL HL HL pop 100 0 1 00 14 15 15 15 pop 100 0 1 00 14 15 15 15 2E Set Up Reaction Setup Reaction Setup Help u Q ai For TE eee sie eee rb eel fusing Usain di then fra plies ite bad a fd ida cin 1 pe pis Ag cs ardent ada the POR reactions excess reaction volume component concentrations and or stock concentrations Clic rin eaction 9 Reaction Mix Calculations Sample Dilution Calculations 3 Diluted Sample Concentration 10X for Reaction Mix 6 6 ng L x Print Reaction Setup sampie Name Stock Concentration ng uL Sample Volume uL Diluent Volume uL Total Volume of Diluted Sample uL 4 pop 100 0 1 00 14 15 15 15 J Notes 34 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Cha
52. Delta status SYBR Green PCR reaction components that consist of two primers designed to amplify the target and reagents SYBR Green dye to detect double stranded DNA Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 119 Experiments Glossary system dye TaqMan reagents target target color Target Library task technical replicates 120 Dye supplied by Applied Biosystems and precalibrated on the 7500 7500 Fast system Before you use system dyes in your experiments make sure the system dye calibration is current in the Instrument Maintenance Manager System dyes of the 7500 7500 Fast system include e FAM dye e JOE dye e NED dye e ROX dye e SYBR Green dye e TAMRA dye e VIC dye e CY3 dye e CY5 dye e TEXAS RED dye PCR reaction components that consist of primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target The nucleic acid sequence that you want to amplify and detect In the 7500 7500 Fast system software a color assigned to a target to identify the target in the plate layout and analysis plots In the 7500 7500 Fast system software a collection of targets to add to experiments The targets in the library contain the target name reporter quencher and target color The target in the library may also contain comments about the target In the 7500 7500 Fast system software the type
53. Experiments regression line reject well relative standard curve method replicate group replicates reporter reverse primer reverse transcriptase Rn ROX dye rs number run method sample Sample DNA 10X Glossary In standard curve and relative standard curve experiments the best fit line from the standard curve Regression line formula Cr m log Qty b where m 1s the slope b is the y intercept and Qty is the standard quantity See also regression coefficients An action that the software performs during analysis to remove one or more wells from further analysis if a specific flag is applied to the well Rejected wells contain results calculated up to the point of rejection Method for determining relative target quantity in samples With the relative standard curve method the 7500 7500 Fast system software measures amplification of the target and of the endogenous control in samples in a reference sample and 1n a standard dilution series Measurements are normalized using the endogenous control Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates target quantity in the samples and in the reference sample The software determines the relative quantity of target in each sample by comparing target quantity in each sample to target quantity in the reference sample A set of identical reactions in an experiment See tech
54. FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA CT 30 982454 Undetermi Undetermi Undetermi 28 280773 28 241549 28 259768 27 241302 27 18564 27 174326 26 389278 26 213354 26 242676 27 36778 27 258842 27 206913 28 325405 28 28138 28 69632 29 64913 29 546368 29 421392 30 557467 30 551697 CT Mean 30 697205 28 260696 28 260696 28 260696 27 200424 27 200424 27 200424 26 281769 26 281769 26 281769 27 277845 27 277845 27 277845 28 434366 28 434366 28 434366 29 538963 29 538963 29 538963 30 697205 30 697205 CT SD 0 247 0 02 0 02 0 02 0 036 0 036 0 036 0 094 0 094 0 094 0 082 0 082 0 082 0 228 0 228 0 228 0 114 0 114 0 114 0 247 0 247 Quantity 625 2 772 365 2 841 162 2 808 998 5 308 388 5 496 287 5 535 287 10 000 10 000 10 000 5 000 5 000 5 000 2 500 2 500 2 500 1 250 1 250 1 250 625 625 the C values are within the expected range gt 8 and lt 35 View Well Table Group By Select Wells With Select Item v i Select Item Expand All Quantity 2 807 509 2 807 509 2 807 509 5 446 654 5 446 654 5 446 654 Quantity 34 422 34 422 34 422 121 319 121 319 121 319 Collapse All on On fF wr 10 11 12 13 14 15 16 u
55. Fast Real Time PCR System Getting Started Guide for Standard Curve 121 Experiments Glossary 122 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Numerics I step RT PCR 8 23 24 31 36 2 step RT PCR 8 23 24 7500 7500 Fast system consumables 4 data collection 2 filters 3 95 reagents 9 A Advanced Setup 10 11 23 29 53 98 alternate experiment workflows See workflows amplification efficiency 27 73 Amplification Plot screen monitor during arun 62 view afterarun 74 amplification plot typical 78 AMPNC flag 89 analysis screens Amplification Plot screen 74 Multicomponent Plot screen 92 Multiple Plots screen 70 navigation tips 69 QC Summary screen 88 Raw Data Plot screen 94 Standard Curve screen 71 Well Table 81 analysis settings advanced 87 baseline 87 CT 87 flag 87 threshold 87 view 86 analyze experiment analyze 68 for more information 73 80 83 87 89 91 94 95 guidelines 69 73 78 83 87 89 91 94 95 omit wells 90 publish the data 83 view Amplification Plot screen 74 view analysis settings 86 view Multicomponent Plot screen 92 view Multiple Plots screen 70 view QC Summary screen 88 view Raw Data Plot screen 94 view Standard Curve screen 71 view Well Table 81 workflow 66 Index Applied Biosystems contacting xi customer feedback on documentation xi Technical Support xi Applied Biosystems 7500 7500 Fast Real Time PCR Syst
56. Getting Started Guide Applied Biosystems 7500 7500 Fast Real Time PCR System Standard Curve Experiments AS Applied Biosystems Getting Started Guide 4 Applied KS Biosystems Applied Biosystems 7500 7500 Fast Real Time PCR System Standard Curve Experiments Get Started Design the Standard Curve Experiment Prepare the Reactions Run the Experiment Analyze the Experiment Copyright 2007 2010 Applied Biosystems All rights reserved Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF For Research Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER Label License The 7500 7500 Fast Real Time PCR System is covered by US patents and corresponding claims in their non US counterparts owned by Applied Biosys tems No right is conveyed expressly by implication or by estoppel under any
57. LE na eet eee xvii Chemical Waste Safety cccccccccc eee tenes X X ICCC Al SACI aus d Gas dete eee ee oe ee Deedee eae ees E Bee XX Physical Hazard Salley sw2 ecetecrviwnchese beer seme td rp ida a aa ems XX Biological Hazard Safety 0 ce ee eee ees xxi Workstation Safety n 0 0 ccc eee ee eee ees xxi Safety and Electromagnetic Compatibility EMC Standards xxii Chapter 1 Get Started 2c04505 eu aueteenee gi SN SS a 1 About the 7500 7500 Fast System 2 eee 2 Supported Consumables aaea eee ee eens 4 About Standard Curve Experiments 2 0 00 cece eee eee ees 7 How to Use This Guide 0 cc eee eee 11 About the Example Standard Curve Experiment 0 000 eee eee eee 12 Example Experiment Workflow 000 eee ees 14 Chapter 2 Design the Standard Curve Experiment 17 Chanter OVEIVICW an pes ro P ee RES DD TIC hetero O ween 18 Create a New Experiment ccccccccccc eee eee eee 19 Define the Experiment Properties 0 0 ccc e 20 Define the Methods and Materials iciccicccicccccc ee eee 22 Set Up the Targets ccccccccc eee teens 24 Set Up the Standards cccccccc eee e es 26 De the SAMPIOS Ses spe ee cae dra ema ARTS A An eee a tee ees 28 Set Up the Run Method 0 0 0 eee ees 30 Review the Reaction Setup 0 cee ee ete eee 32 Order Materials for the Experiment 0000 cee eee ees 3 App
58. NG Hot Replace lamp with an Applied Biosystems lamp AVERTISSEMENT Composants br lants Remplacer la lampe par une lampe Applied Biosystems CAUTION Hot surface ATTENTION Surface br lante DANGER High voltage shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel DANGER Haute tension WARNING To reduce the chance of electrical AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont l ouverture n cessite l utilisation d outils L instrument ne contient aucune piece r parable par utilisateur Toute intervention doit tre effectu e par le personnel de service qualifi de Applied Biosystems CAUTION Moving parts ATTENTION Parties mobiles WARNING This instrument is designed for 12 V 75 W Halogen lamps only AVERTISSEMENT Cet instrument est con u pour des lampes d halog ne de 12 V et 75 W seulement XIV Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface Safety Labels on Instruments Locations of The Applied Biosystems 7500 7500 Fast Real Time PCR System contains warnings at Warnings the locations shown below Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve XV Experiments Preface General Instrument Safety Genera
59. PERA PES LATTES DADE RES EG 83 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 67 Experiments Chapter 5 Analyze the Experiment Analyze the Experiment Analyze the Experiment The 7500 software analyzes the experiment and displays results in the analysis screens for example the Amplification Plot screen QC Summary screen and so on About the For the standard curve example experiment use the data file that is installed with the Example 7500 software The data file was created with the same design parameters that are Experiment provided in Chapter 2 then run and analyzed on a 7500 7500 Fast instrument You can find the data file for the example experiment on your computer at lt drive gt Applied Biosystems lt software name gt experiments Standard Curve Example eds where e lt drive gt is the computer hard drive on which the 7500 software is installed e lt software name gt is the current version of the 7500 software Analyze the 1 Double click 3 7500 software or select Start All Programs gt Applied Example Biosystems gt 7500 Software gt lt software name gt Experiment where lt software name gt is the current version of the 7500 software 2 In the Home screen click Open 3 In the Open dialog box navigate to the experiments folder at lt drive gt Applied Biosystems lt software name gt experiments 4 Double click Standard Curve Ex
60. RA EH Fam Wells E 6 Unknown E 15 Standard 1 3 Negative Control Notes 72 Empty Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments 93 nr Chapter 5 Analyze the Experiment View the Raw Data Plot Analysis When you analyze your own standard curve experiment look for Guidelines e Passive reference The passive reference dye fluorescence level should remain relatively constant throughout the PCR process e Reporter dye The reporter dye fluorescence level should display a flat region corresponding to the baseline followed by a rapid rise in fluorescence as the amplification proceeds e Any irregularities in the signal There should be no spikes dips and or sudden changes in fluorescence e Negative control wells There should be no amplification in the negative control wells For More For more information on the Multicomponent Plot screen open the 7500 Software Help Information by clicking or pressing F1 View the Raw Data Plot The Raw Data Plot screen displays the raw fluorescence not normalized for each optical filter for the selected wells during each cycle of the real time PCR About the In the standard curve example experiment you review the Raw Data Plot screen for a Example stable increase in signal no abrupt changes or dips from the appropriate filter Experiment Raw Data Plot View the Raw 1 Inthe nav
61. Regression coefficient calculated from the regression line in the standard curve The slope indicates the PCR amplification efficiency for the assay A slope of 3 32 indicates 100 amplification efficiency See also amplification efficiency EFF and regression line Abbreviation for single nucleotide polymorphism The SNP can consist of a base difference or an insertion or deletion of one base Used in genotyping experiments a PCR reaction that contains primers to amplify the SNP and two probes to detect different alleles In the 7500 7500 Fast system software a collection of SNP assays to add to genotyping experiments The SNP assays in the library contain the SNP assay name SNP assay color and for each allele the allele name or base s reporter quencher and allele colors The SNP assays 1n the library may also contain the assay ID and comments about the SNP assay Type of 7500 7500 Fast system calibration in which the system maps the positions of the wells in the sample block ROI calibration data are used so that the software can associate increases in fluorescence during a run with specific wells in the reaction plate In the thermal profile a group of one or more steps There are three types of stages holding stage including pre PCR read and post PCR read cycling stage also called amplification stage and melt curve stage Sample that contains known standard quantities Standard reactions are used in quantitation experimen
62. Run Method perature Monitor the Run You can view the progress of the run in real time as described below During the run periodically view all three available plots from the 7500 software for potential problems To Action Stop the run 1 In the 7500 software click STOP RUN 2 In the Stop Run dialog box click one of the following Stop Immediately to stop the run immediately Stop after Current Cycle Hold to stop the run after the current cycle or hold Cancel to continue the run View amplification data Select By Amplification Plot in real time See About the Amplification Plot Screen on page 62 View progress of the Select Run Method ea a See About the Run Method Screen on page 63 screen Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 61 Experiments Chapter 4 Run the Experiment Monitor the Run To Action Enable disable the Select or deselect Enable Notifications POUCA ono ends See Enable the Notification Settings Optional on page 59 About the Amplification Plot Screen The screen displays sample amplification as your instrument collects fluorescence data during a run If a method is set up to collect real time data the Amplification Plot screen displays the data for the wells selected in the View Plate Layout tab The plot displays norm
63. alized dye fluorescence ARn as a function of cycle number The figure below shows the Amplification Plot screen as it appears during the example experiment To view data in the Amplification Plot screen select the wells that you want to view in the View Plate Layout tab Amplification Plot lt View Plate Layout gt Select Wells With Select Item P Pa REE View Legend Amplification Plot 11 12 pop pop1 pop1 pop2 pop2 pop2 gm jm m fo jo jo jo ju ju 0 001 0 0001 0 00001 0 000001 dj T t T T T T T T T r T re r T 2 4 6 86 0 12 14 6 6 3 2 N 7 BHD RK ABB HA Cycle The Amplification Plot screen is useful for identifying and examining abnormal amplification Abnormal amplification can include e Increased fluorescence in negative control wells e Absence of detectable fluorescence at an expected cycle determined from previous similar experiments that were run using the same reagents under the same conditions If you notice abnormal amplification or a complete absence of florescence troubleshoot the error as explained in the 7500 Software Help click or press F1 Notes 62 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 4 Run the Experiment AL Monitor the Run About the Run Method Screen The screen displays the run method selected for the run 1n progress The sof
64. ample eds to open the example experiment data file Note The experiments folder contains several data files be sure to select Standard Curve Example eds 5 In the navigation pane click Analysis The 7500 software analyzes the data using the default analysis settings See Navigation Tips on page 69 for information on navigating within the analysis screens File Edit Instrument Analysis Tools Help New Experiment 29 Open pj Save EF Close SB Export E PrintReport Experiment Menu X Experim standard curve example experime Ty Standard Curvi Reage TaqMan Reagents EADE Analysis Settings Amplification Plot lt View Plate Layout Plot Settings Plot Type ARn vs Cycle 4 Graph Type Log PlotColor Well w 5 Save current settings as the default mem P P 8 Em z4 2 3 4 5 6 7 8 9 ch ex er 278 3 3 Ste Sh Su Sta biardi Cite 10 Amplification Plot Es To ee To To TT Tm Tm A Notes 68 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment Analyze the Experiment Guidelines When you analyze your own standard curve experiment e Immediately after a run the 7500 software automatically analyzes the data using the default analysis settings then displays the Amplification Plot screen on your computer e To reanalyze the data select all the wells
65. an Gene Expression Assays Protocol 4334429 Primer Express Software Version 3 0 Getting Started Guide 4362460 TaqMan Gene Expression Assays Protocol 4333458 User Bulletin 2 Relative Quantitation of Gene Expression 4303859 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface How to Obtain More Information Documents Related to Standard Curve Experiments Document PN Amplification Efficiency of TagMan Gene Expression Assays Application Note 127AP05 Custom TaqMan Gene Expression Assays Protocol 4334429 Primer Express Software Version 3 0 Getting Started Guide 4362460 TaqMan Gene Expression Assays Protocol 4333458 User Bulletin 2 Relative Quantitation of Gene Expression 4303859 Documents Related to the Reagent Guide Document PN Applied Biosystems High Capacity cDNA Reverse Transcription Kits Protocol 4375575 Custom TaqMan Gene Expression Assays Protocol 4334429 Custom TaqMan Genomic Assays Protocol Submission Guidelines 4367671 Custom TaqMan SNP Genotyping Assays Protocol 4334431 Power SYBR Green PCR Master Mix and RT PCR Protocol 4367218 Pre Developed TaqMan Assay Reagents Allelic Discrimination Protocol 4312214 Primer Express Software Version 3 0 Getting Started Guide 4362460 SYBR Green PCR and RT PCR Reagents Protocol 4304965 SYBR Green PCR Maste
66. andard curve experiment look for the following in each Guidelines filter e Characteristic signal growth e No abrupt changes or dips For More For more information on the Raw Data Plot screen open the 7500 Software Help by Information clicking or pressing F1 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 95 Experiments Chapter 5 Analyze the Experiment a View the Raw Data Plot Notes 96 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Appendix A N Alternate Experiment Workflows This appendix covers E Advanced Setup Workflow 0 0 0 0 00 00 ccs 98 E QuickStart Workflow ao hn 65 da fadada ROSE ERIE 98 ES EE ER 100 E Template WOW rs po pads PD Sp SR de OEE pi a da qa add 102 E Export Import Workflow ccccccccccc nes 104 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500 Software Help Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 97 Experiments A Appendix A Alternate Experiment Workflows Advanced Setup Workflow Advanced Setup Workflow When you create an experiment using Advanced Setup in the 75
67. art Workflow When you create an experiment using QuickStart you can run the reactions on the instrument with no reaction plate setup information 1 Prepare the PCR reactions Experiment Type Prepare the Relative standard curve Template Sample dilutions Standard dilution series Reaction mix Reaction plate Standard curve oOQando Comparative Cr a Template b Sample dilutions Genotyping P e c Reaction mix Presence absence d Reaction plate 2 QuickStart the experiment a Double click 3 7500 software or select Start All Programs gt Applied Biosystems gt 7500 Software gt lt software name gt where lt software name gt is the current version of the 7500 software b In the Home screen click Q QuickStart c Select the Experiment Properties tab default enter the experiment name then select the experiment properties d Select the Run Method tab review the reaction volume and thermal profile then edit as needed 3 Run the experiment IMPORTANT While the 7500 7500 Fast instrument is performing a run do not create experiments perform maintenance or allow the computer to run antivirus software or to enter hibernation Performing such activities while the instrument is running an experiment will cause gaps in data collection Load the reaction plate into the instrument a b Start the run c Optional Monitor the ru
68. ast Reaction 8 tube strips can be used only on the 7500 Fast system For the 7500 system use MicroAmp Optical 8 Tube Strips Reaction tube strips Place the tube strips in the PPH for tube strips Note Fast Reaction tubes cannot be used in the 7500 Fast system Reaction tubes Place the tubes in the PPH Note MicroAmp Fast Reaction Tubes PN 4358297 cannot be used in the 7500 Fast system IMPORTANT For optimal performance with partial loads For a 7500 Instrument Load at least 16 tubes arranging them first in the center columns of the instrument system columns 6 and 7 Move outward towards columns 1 and 12 as you add more tubes For a 7500 Fast Instrument e Place empty tube strips in columns 1 and 12 to prevent crushing of tubes containing samples e Place tube strips with samples in the PPH vertically starting in columns 6 and 7 and moving outward e A maximum of 6 tube strips can be used in the 7500 Fast instrument Leave columns 2 3 10 and 11 empty 4 Close the tray door Apply pressure to the right side of the tray and at an angle 58 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 4 Run the Experiment A Enable the Notification Settings Optional Enable the Notification Settings Optional About the Example Experiment Set Up the Notification Settings Notes Enable the notification settings
69. ational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov Workstation Safety Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions AM caution MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks e Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse e Position the keyboard mouse and monitor to promote relaxed body and head postures Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve xxi Experiments Preface Safety and Electromagnetic Compatibility EMC Standards Safety and Electromagnetic Compatibility EMC Standards U S and Canadian Safety Sta
70. bat Reader f Desktop Netscape Microsoft Macintosh Adobe Operating Naviaator Internet Safari Acrobat System g Explorer Reader Windows v6 x or later v6 x or later Not applicable v4 0 or later 2000 NT XP Vista Macintosh Not supported Not supported v2 0 4 or later v4 0 or later OS 9 or later IMPORTANT Make sure that cookies and Java Script are turned on e To find your assay in the Applied Biosystems Store complete the Find Assay pane a Click the Enter Gene Name field enter the gene name then click Find Assay b In the Find Assay Results dialog box select your assay c Click Apply Assay Selection Find Assay Results 7 Assays Found Click the Assay ID to view the assay in the Applied Biosystems Store Select an assay to detect and quantify the target sequence rnase p Information Availability Made to Order Made to Order Made to Order Made to Order Made to Order Made to Order Made to Order Assay ID Dm01837058 q1 Dm01837057 a1 Dm01837056 q1 Dm01837055 q1 Rn01479852 m1 Rn01479850 mt Rn01479855 m1 Gene Symbol Rpp30 Rpp30 Rpp30 Rpp30 LOC685332 rCG48258 LOC685332 rCG48258 LOC685332 rCG48258 Gene Name RNaseP protein p30 RNaseP protein p30 RNaseP protein p30 RNaseP protein p30 similar to Ribonuclease P protein subunit p30 RNaseP protein p30 RNas Gene rCG48258 Celera Annotation similar to Ribonuclease P protein subunit
71. cDNA complementary DNA RNA ou are adding purified gDNA to the real time PCR reactions You have already extracted the gDNA from tissue or siimple Notes 22 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment Define the Methods and Materials Design When you design your own standard curve experiment Guidelines e Select Standard Curve as the quantitation method The standard curve method is used to determine the absolute target quantity in samples When setting up your reaction plate the standard curve method requires targets standards and samples e Select the reagents you want to use Select TaqMan Reagents if you want to use TaqMan reagents to detect amplification and quantify the amount of target in the samples TaqMan reagents consist of two primers and a TaqMan probe The primers are designed to amplify the target The TaqMan probe is designed to hybridize to the target and generate fluorescence when the target is amplified Select SYBR Green Reagents if you want to use SYBR Green reagents to detect amplification and quantify the amount of target in the samples SYBR Green reagents consist of two primers and SYBR Green dye The primers are designed to amplify the target The SYBR Green dye generates fluorescence when it binds to double stranded DNA SYBR Green dye is often part of the SYBR Green
72. ckslash greater than sign gt less than sign lt asterisk question mark quotation mark vertical line colon or semicolon The type of experiment you are performing using the 7500 7500 Fast system e Standard curve e Comparative Cy AAC e Relative standard curve e Melt curve not available in the Design Wizard e Genotyping e Presence absence The experiment type you select affects the setup run and analysis Oligonucleotide that flanks the 5 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target In the thermal profile a stage that includes one or more steps You can add a holding stage to the thermal profile to activate enzymes to inactivate enzymes or to incubate a reaction A gene that is involved in basic cellular functions and is constitutively expressed Housekeeping genes can be used as endogenous controls See also endogenous control In presence absence experiments a short synthetic DNA template that is added to PCR reactions You can use the IPC to distinguish between true negative results that is the target is absent in the samples and negative results caused by PCR inhibitors incorrect assay setup or reagent or instrument failure TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assays that have been previously manufactured passed quality control specifications and stored in inventory In
73. condary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste Disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal
74. ctions for standard curve experiments Sample The sample in which the quantity of the target 1s unknown Standard A sample that contains known standard quantities used in quantitation experiments to generate standard curves Standard dilution series A set of standards containing a range of known quantities The standard dilution series is prepared by serially diluting standards Replicates The total number of identical reactions containing identical samples components and volumes Negative Controls Wells that contain water or buffer instead of sample template No amplification of the target should occur in negative control wells Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 1 Experiments 4 Chapter 1 Get Started About Standard Curve Experiments PCR Options When performing real time PCR select between e Singleplex and multiplex PCR below and e I step and 2 step RT PCR page 8 Singleplex vs Multiplex PCR You can perform a PCR reaction using either e Singleplex PCR In singleplex PCR a single primer set is present in the reaction tube or well Only one target or endogenous control can be amplified per reaction or e Multiplex PCR In multiplex PCR two or more primer sets are present in the reaction tube or well Each set amplifies a specific target or endogenous control Typically a probe labeled with FAM dye detects the target and a pr
75. d see View the Analysis Settings on page 86 For More For more information on the Amplification Plot screen open the 7500 Software Help by Information clicking or pressing F1 Notes 80 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments View the Well Table The Well Table displays data for each well in the reaction plate including e The sample name target name task and dyes e The calculated threshold cycle C normalized fluorescence Rn and quantity values e Comments e Flags About the Example e Quantity values Experiment View the Well 1 Table 2a e Flags Chapter 5 Analyze the Experiment View the Well Table e C values including Cy standard deviation Note If no data are displayed click Analyze In the standard curve example experiment you review the Well Table for In the navigation pane select Analysis then select the View Well Table tab 2 Use the Group By drop down list to group wells by a specific category For the example experiment group the wells by replicate flag or Cy value Note You can select only one category at a time a In the Group By drop down list select Replicate The software groups the replicate wells negative controls standards and samples In the example experiment note that the quantity values within each replicate group are similar Note To move a column drag the column head
76. d as is and closed Note For the example experiment do not perform the run at this time Notes 40 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment Fo Finish the Design Wizard Finish the 1 Atthe bottom of the 7500 software screen click Finish Designing Experiment Design Wizard In the Review Plate Layout for Experiment window review the plate layout Make sure there are e 6 Unknown wells ij e 15 Standard wells E e 3 Negative control wells 1 e 72 Empty wells Note If the plate layout is incorrect click Return to the Wizard and check your entered values 3 Click Save Experiment Review Plate Layout for Experiment Standard Curve Example E Review the plate layout then select what you want to do next 3 Save Experiment Start Run for This Experiment Edit Plate Layout Ces Another Experiment Return to the Wizard Using the Design Wizard Save and close this Save this experiment then Use Advanced Setup to edit Save and close this Continue designing this experiment start the run Make sure the the plate layout experiment then create experiment using the Design reaction plate is loaded into another experiment using the Wizard the instrument Design Wizard pop1 pop2 pop2 pop2 g RNase g RNase g RNase mae m e m e mae E E acl g RNase 5 RNas
77. dards For example the standard stock is used to prepare the first dilution point the first dilution point is used to prepare the second dilution point and so on In the 7500 7500 Fast system software the volumes needed to prepare a standard dilution series are calculated by the number of dilution points the number of standard replicates the starting quantity the serial factor and the standard concentration in the stock See also standard curve A known quantity in the PCR reaction e In standard curve experiments the quantity of target in the standard In the 7500 7500 Fast system software the units for standard quantity can be for mass copy number viral load or other units for measuring the quantity of target e In relative standard curve experiments a known quantity in the standard Standard quantity can refer to the quantity of cDNA or the quantity of standard stock in the PCR reaction The units are not relevant for relative standard curve experiments because they cancel out in the calculations When defining a standard curve 1n the 7500 7500 Fast system software corresponds to the highest or lowest quantity step A component of the thermal profile For each step in the thermal profile you can set the ramp rate ramp increment for melt curve steps hold temperature hold time duration and you can turn data collection on or off for the ramp or the hold parts of the step For cycling stages a step is also defined by the Auto
78. dbiosystems com IMPORTANT The e mail address above is only for submitting comments and suggestions relating to documentation To order documents download PDF files or for help with a technical question go to http www appliedbiosystems com then click the link for Support See How to Obtain Support on page x1 How to Obtain Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support screen you can Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents e Obtain information about customer training Download software updates and patches In addition the Support screen provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities IMPORTANT When directed to do so by this guide or when you need to schedule maintenance for your 7500 7500 Fast instrument such as annual planned maintenance or temperature verification calibration contact the Applied Biosystems Care Center To obtain a phone number for or to send an e mail to the center go to http www appliedbiosystems com support contact Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve x
79. dle the halogen lamp AN ANN The lamp is extremely hot Do not touch the lamp until it has cooled to room temperature Nore CHEMICAL HAZARD Ethanol is a flammable liquid and vapor Exposure causes eye skin and respiratory tract irritation and may cause central nervous system depression and liver damage Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves JN Uledi ELECTRICAL HAZARD Failure to ground the instrument properly can lead to an electrical shock Ground the instrument according to the provided instructions Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Symbols on Instruments Preface Symbols on Instruments Electrical The following table describes the electrical symbols that may be displayed on Symbols on Applied Biosystems instruments Instruments Symbol Description Symbol Description Indicates the On position of the Indicates a terminal that may be main power switch cm connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates the Off position of the Indicates a protective grounding main power switch terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a standby switch by Indicates a terminal that can which the instrument
80. e 5 RNase g RNase g RNase g RNase g RNase g RNase g RNase g RNase g RNase g RNase EC EC9 EC 9EC9 9EC9 72609 4 EC 4 RC 4 9EC9 RIE RIE RIR aa 4 Inthe Save Experiment dialog box enter Standard Curve Example Setup eds in the File name drop down list then click Save The example experiment is saved and closed and you are returned to the Home screen IMPORTANT Do not save the experiment using the default file name Doing so will overwrite the example experiment already present in the experiments folder Note By default the example experiment is saved to the lt drive gt Applied Biosystems lt software name gt experiments folder Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 41 Experiments Chapter 2 Design the Standard Curve Experiment Finish the Design Wizard Design When you design your own standard curve experiment Guidelines e In the Review Plate Layout for Experiment window select the appropriate exit option Click To Save Experiment Save and close the experiment without making any further changes or starting the run Start Run for This Experiment Save the experiment and start the run Make sure the reaction plate is loaded in the instrument Edit Plate Layout Use Advanced Setup to edit the plate layout Create Another Experiment Save and close the current experiment then
81. e Include all required components e Prepare the reagents according to the manufacturer s instructions e Keep the assay mix protected from light in the freezer until you are ready to use it Excessive exposure to light may affect the fluorescent probes e Prior to use Mix the master mix thoroughly by swirling the bottle Resuspend the assay mix by vortexing then centrifuge the tube briefly Thaw any frozen samples by placing them on ice When thawed resuspend the samples by vortexing then centrifuge the tubes briefly For More For more information on preparing the reaction mix refer to the protocol appropriate for Information the reagents you are using in the PCR reactions TaqMan Gene Expression Assays Protocol Custom TagMan Gene Expression Assays Protocol Notes 50 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 3 Prepare the Reactions a Prepare the Reaction Plate Prepare the Reaction Plate Prepare the reactions for each replicate group then transfer them to the reaction plate using the plate layout displayed in the 7500 software About the For the standard curve example experiment Example e A MicroAmp Optical 96 Well Reaction Plate is used Experiment p The reaction volume is 25 uL well e The reaction plate contains 6 Unknown wells 1 15 Standard wells E 3 Negative control we
82. e diluted samples on ice until you prepare the reaction plate Preparation When you prepare your own standard curve experiment Guidelines Sample dilutions may be necessary because the sample volume is limited to 10 of the total reaction volume in the 7500 software You must perform the sample dilutions before adding the samples to the final reaction mix For optimal performance of TagMan Gene Expression Assays or Custom TaqMan Gene Expression Assays use 10 to 100 ng of cDNA template per 20 uL reaction For Fast reagents Applied Biosystems recommends 10 ng e Use TE buffer or water to dilute the sample For More For more information on Applied Biosystems assays refer to the Information TaqMan Gene Expression Assays Protocol Custom TaqMan Gene Expression Assays Protocol Notes 46 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 3 Prepare the Reactions iain Prepare the Standard Dilution Series Prepare the Standard Dilution Series Prepare the standard dilution series using the volumes that were calculated by the 7500 software Complete the Reaction Mix Calculations Tab see page 33 on page 32 About the For the standard curve example experiment Example e The standard concentration stock is 20 000 copies uL Experiment The volumes calculated in the software are Dilution ence Source Volume Diluent Volu
83. e during PCR O o Step 2 Denaturation cycles o When the DNA is denatured into gt gt gt gt single stranded DNA the Advantages SYBR Green dye is released and e Economical no probe needed the fluorescence is drastically reduced e Allows for melt curve analysis to measure pe Step 3 Polymerization the Tm of all PCR products e During extension primers e Canbeusedforeither1 or2 stepRTPCR O Hi anneal and PCR product O o 0 is generated Limitations pane Binds nonspecifically to all double stranded no DNA sequences To avoid false positive oe e e Step E ld completed signals check for nonspecific product 35 SYBR Green dye binds to the ESA A a A DRL double stranded product resulting in a net increase in fluorescence detected by the instrument formation using melt curve or gel analysis Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 9 Experiments ao Chapter 1 Get Started About Standard Curve Experiments Other Reagents If you use fluorescence based reagents other than TaqMan reagents you must design your experiment using Advanced Setup instead of the Design Wizard see Advanced Setup Workflow on page 98 For More For more information on real time PCR experiments PCR options and reagents refer to Information the Applied Biosystems Real Time PCR Systems Reagent Guide Notes 10 Applied Biosystems
84. e edge of the film in ap place grasp one end of the end tab and pull up and away A gt sharply Repeat for the other end tab gt Repeat step f to ensure a tight evaporation free seal While applying firm pressure run the edge of the applicator along all four sides of the outside border of the film Note Optical adhesive films do not adhere on contact The films require the application of pressure to ensure a tight seal Inspect the reaction plate to be sure all wells are sealed You should see an imprint of all wells on the surface of the film For More For more information on Information e Preparing the reaction plate Refer to the protocol appropriate for the reagents you are using in the PCR reactions TaqMan Gene Expression Assays Protocol Custom TagMan Gene Expression Assays Protocol e Consumables See Supported Consumables on page 4 Using Advanced Setup to change the plate layout See page 98 Notes 54 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 4 Run the Experiment This chapter covers E Cae DEDO oko oh a hdd EOE RESS dA SR ad Dad dA DS Ad S 56 re es dadrsns oes ESAEREN ER Pr CL pena dd E Enable the Notification Settings Optional 0 00 e eee eee 59 nee rsss siso Tia eee eens O SD E esse ee es 61 a oo ee ho ee GALP TAS PERA dA 61 lek hk ce oy adere ed Eddie SA DA RES
85. e experiment there are no outliers for RNase P 6a Amplification Plot Plot Settings Plot Type Cr vs Well v Graph Type Plot Color Save current settings as Notes Legend Mia De Mic Mv WE MF Mic MH the default PPawBHE Amplification Plot 5 10 15 20 Well Number Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 17 Experiments Chapter 5 Analyze the Experiment View the Amplification Plot Analysis When you analyze your own standard curve experiment look for Guidelines Outliers e A typical amplification plot The 7500 software automatically calculates baseline and threshold values based on the assumption that the data exhibit a typical amplification plot A typical amplification plot has four distinct sections a Plateau phase b Linear phase c Exponential geometric phase d Baseline 0 0001 IMPORTANT Experimental error such as contamination or pipetting errors can produce atypical amplification curves that can result in incorrect baseline and threshold value calculations by the 7500 software Therefore Applied Biosystems recommends that you examine the Amplification Plot screen and review the assigned baseline and threshold values for each well after analysis is complete e Correct baseline and threshold values See the threshold examples on page 79 and the baseline examples on page 80 N
86. e experiments fluorescence data collected during the post PCR read are displayed in the presence absence plot and used to make detection calls Also called endpoint read Used in genotyping and presence absence experiments the part of the instrument run that occurs before amplification The pre PCR read is optional but recommended Fluorescence data collected during the pre PCR read can be used to normalize fluorescence data collected during the post PCR read PCR reaction component that contains the forward primer and reverse primer designed to amplify the target PCR reaction component that contains the primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target See custom dye and system dye In quantitation experiments the method used to determine the quantity of target in the samples In 7500 7500 Fast systems there are three types of quantitation methods standard curve relative standard curve and comparative C AAC In quantitation experiments the amount of target in the samples Absolute quantity can refer to copy number mass molarity or viral load Relative quantity refers to the fold difference between normalized quantity of target in the sample and normalized quantity of target in the reference sample A molecule attached to the 3 end of TaqMan probes to prevent the reporter from emitting fluorescence while the probe is intact With TaqMan reagents a nonfluorescent
87. e information on Information Completing the Methods amp Materials screen Open the 7500 Software Help by clicking or pressing F1 Using Advanced Setup See Advanced Setup Workflow on page 98 Using other quantitation methods Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR Systems Getting Started Guide for Relative Standard Curve and Comparative Cr Experiments TaqMan and SYBR Green reagents Refer to the Applied Biosystems Real Time PCR Systems Reagent Guide PCR including singleplex vs multiplex PCR and 1 step vs 2 step RT PCR Refer to the Applied Biosystems Real Time PCR Systems Reagent Guide Set Up the Targets About the Example Experiment Complete the Targets Screen Notes In the Targets screen you enter the number of targets you want to quantify in the PCR reaction plate then set up the experiments for each target In the standard curve example experiment One target is quantified in the reaction plate The Set Up Standards check box is selected When this check box is selected the software automatically displays the Standards screen after you complete the Targets screen In the Standards screen you can set up a standard curve for the target experiment see Set Up the Standards on page 26 The Target 1 experiment is set up for the target you are studying For the example experiment the target is the RNase P gene Click the How many targets do you want to quan
88. e name gt Experiment where lt software name gt is the current version of the 7500 software 2 Inthe Home screen click Open 3 In the Open dialog box navigate to the experiments folder default lt drive gt Applied Biosystems lt software name gt experiments 4 Double click Standard Curve Example Setup eds to open the example experiment file that you created in Chapter 2 Load the PAIN WARNING PHYSICAL INJURY HAZARD During operation the sample Reaction Plate block can be heated to 100 C Before performing the following procedure be sure to Into the wait until the sample block reaches room temperature Instrument IMPORTANT Wear powder free gloves when you handle the reaction plate 1 Push the tray door to open 1t 2 Load the plate into the plate holder in the instrument Ensure that the plate is properly aligned in the holder 7500 system 7500 Fast system Load standard plates with Load Fast plates with the the notched A12 position at notched A1 position at the the top right of the tray top left of the tray Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 57 Experiments A Chapter 4 Run the Experiment Notes Prepare for the Run 3 Place the reactions in the precision plate holder PPH If you use e A reaction plate Place the reaction plate in the PPH with well Al at the back left corner Note F
89. em See 7500 7500 Fast system assumptions for using this guide viii B BADROX flag 89 baseline correct values 78 examples 80 manually adjust 87 biohazardous waste handling xix biological hazard guidelines xxi BLFAIL flag 89 C CAUTION description xii chemical safety xvii xviii chemical waste safety xix consumables Also see materials required 4 supported 4 conventions safety xii conventions used in this guide viii create new experiment 19 CT 83 CTFAIL flag 89 Custom assays 36 customer feedback on Applied Biosystems documents xi D DANGER description xii data about data collection 2 example experiment 11 68 publish 83 design experiment create new 19 define experiment properties 20 define methods and materials 22 finish Design Wizard 40 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 123 Experiments Index for more information 21 24 25 27 30 31 36 39 42 guidelines 21 23 25 27 29 31 36 39 42 order materials 37 review reaction setup 32 set up run method 30 set up samples 28 set up standards 26 set up targets 24 workflow 18 Design Wizard Experiment Properties screen 20 finish 40 Materials List screen 37 Methods amp Materials screen 22 Reaction Setup screen 32 Run Method screen 30 Samples screen 28 Standards screen 26 Targets screen 24 deviation standard 79 documentation related ix electrical safe
90. ents 4 Chapter 1 Get Started About the 7500 7500 Fast System About the 7500 7500 Fast System The Applied Biosystems 7500 7500 Fast Real Time PCR System is a 96 well five color platform that uses fluorescence based polymerase chain reaction PCR reagents to provide e Quantitative detection of target nucleic acid sequences targets using real time analysis e Qualitative detection of targets using post PCR endpoint analysis e Qualitative analysis of the PCR product achieved by melt curve analysis that occurs post PCR About Data The 7500 7500 Fast system collects raw fluorescence data at different points during a Collection PCR depending on the type of run that the instrument performs Run Type Data Collection Point Real time runs Standard curve The instrument collects data after each extension step of the PCR Relative standard curve Comparative C AAC Post PCR Genotyping The instrument collects data endpoint runs e Before the PCR For presence absence experiments data collection before the PCR is optional but recommended e Optional During the PCR The instrument can collect data during the run real time collecting data during the run can be helpful for troubleshooting e After the PCR Presence absence Regardless of the run type a data collection point or read on the 7500 7500 Fast instrument consists of three phases 1 Excitation The instrument i
91. ep your spine in a good neutral position while lifting with your legs e Participants should coordinate lift and move intentions with each other before lifting and carrying e Instead of lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Operating the Ensure that anyone who operates the instrument has Instrument Received instructions in both general safety practices for laboratories and specific safety practices for the instrument e Read and understood all applicable Material Safety Data Sheets MSDSs See About MSDSs on page xvii Nee PHYSICAL INJURY HAZARD Use this instrument as specified by Applied Biosystems Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Cleaning or AN MNA Before using a cleaning or decontamination method other than those Decontaminating recommended by the manufacturer verify with the manufacturer that the proposed the Instrument method will not damage the equipment xvi Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface Chemical Safety Chemical Safety Chemical Hazard A warnino CHEMICAL HAZARD Before handling any chemicals refer to Warning the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions
92. er 1 zl E TTiyy pay tyr 30 yL 4 FAFU bf ae aeg aug ag age af ti 100 pL I if tb ul ub AT AI ul Ii ty ty troup tbe vi al maximum uU uU U u U U U DO G V F U UF u U maximum reaction reaction volume volume Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments About Standard Curve Experiments Real Time PCR Experiments About Standard Curve Experiments Notes Standard curve experiments are real time PCR experiments In real time PCR Chapter 1 Get Started 4 About Standard Curve Experiments experiments The instrument monitors the progress of the PCR as it occurs Data are collected throughout the PCR process Reactions are characterized by the point in time during cycling when amplification of a target is first detected Note In this guide the term experiment refers to the entire process of performing a run using the 7500 7500 Fast system including setup run and analysis The standard curve method 1s used to determine the absolute target quantity in samples With the standard curve method the 7500 software measures amplification of the target in samples and in a standard dilution series Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates the absolute quantity of target in the samples Components The following components are required when setting up PCR rea
93. ersonnel who schedule manage and perform the tasks required to prepare your site for installation of the 7500 7500 Fast system Guide Purpose and Audience PN Applied Biosystems 7500 7500 Fast Real Explains how to perform experiments on the 7500 7500 Fast 4387784 Time PCR System Getting Started Guide system Each Getting Started Guide functions as both a ore non ping FAPRUTENIS e Tutorial using example experiment data provided with the Applied Biosystems 7500 7500 Fast Real A a 7500 7500 Fast Real Time PCR 4387785 Time PCR System Getting Started Guide aE So ware for Presence Absence Experiments e Guide for your own experiments Intended for laboratory staff and principal investigators who Applied Biosystems 7500 7500 Fast Real l l 4387783 Time PCR System Getting Started Guide perform experiments using the 7500 7500 Fast system for Relative Standard Curve and Comparative C Experiments Applied Biosystems 7500 7500 Fast Real 4387779 Time PCR System Getting Started Guide for Standard Curve Experiments Applied Biosystems 7500 7500 Fast Real Explains how to maintain the 7500 7500 Fast system 4387777 Heo petal Mia enanee Guide Intended for laboratory staff responsible for the maintenance of Applied Biosystems 7500 7500 Fast Real the 7500 7500 Fast system 4387778 Time PCR System Computer Setup Guide Applied Biosystems Real Time PCR Provides information about the reagents you can use on 4387787 Systems Reagent Guide Applied Biosystem
94. etermi 3 A3 O RNase PT NTC FAM TAMRA Undetermi RNase P TAMRA STANDARD 10000 0 4 A10 RNase PT STANDARD FAM TAMRA 26 389278 26 281769 0 094 10 000 5 A11 RNase PT STANDARD FAM TAMRA 26 213354 26 281769 0 094 10 000 6 A12 RNase PT STANDARD FAM TAMRA 26 242676 26 281769 0 094 10 000 RNase P TAMRA STANDARD 1250 0 7 B7 RNase PT STANDARD FAM TAMRA 29 64913 29 538963 0 114 1 250 8 B8 O RNase PT STANDARD FAM TAMRA 29 546368 29 538963 0 114 1 250 9 B9 O RNase P T STANDARD FAM TAMRA 29 421392 29 538963 0 114 1 250 RNase P TAMRA STANDARD 2500 0 10 B4 O RNase PT STANDARD FAM TAMRA 28 325405 28 434366 0 228 2 500 11 B5 O RNase P T STANDARD FAM TAMRA 28 28138 28 434366 0 228 2 500 12 B6 O RNase P T STANDARD FAM TAMRA 28 69632 28 434366 0 228 2 500 RNase P TAMRA STANDARD 5000 0 13 B1 d RNase P T STANDARD FAM TAMRA 27 36778 27 277845 0 082 5 000 14 B2 O RNase PT STANDARD FAM TAMRA 27 258842 27 277845 0 082 5 000 15 B3 O RNase PT STANDARD FAM TAMRA 27 206913 27 277845 0 082 5 000 RNase P TAMRA STANDARD 625 0 5 16 B10 O RNase PT STANDARD FAM TAMRA 30 557467 30 697205 0 247 625 IT B11 RNase PT STANDARD FAM TAMRA 30 551697 30 697205 0 247 625 18 B12 a 4 RNase PT STANDARD FAM TAMRA 30 982454 30 697205 0 247 625 popl RNase P TAMRA UNKNOWN 5 Select the Omit check box next to well B12 Notes 90 Applied Biosystems 7500 7500 Fast Real Time PCR Sys
95. fault Inthe Well Count pane verify that there are e 6 Unknown wells e 15 Standard wells E e 3 Negative control wells 1 e 72 Empty wells In the View Plate Layout tab a In the Arrange Plate by drop down list select Rows default 26 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments 10 Chapter 2 Design the Standard Curve Experiment En Set Up the Samples b In the Place Negative Controls in drop down list select Upper Left default Click Next 2C Set Up Samples Q Instructions Set Up Samples Samples Help Enter the number of samples to test in the reaction plate enter the number of sample target reaction replicates enter the number of negative controls enter sample names associate the samples with biological replicate groups then selectthe sample target reactions to set up Required View Plate Layout a lt gt How many samples do you want to test in the reaction plate Arrange Plate by Rows v Place Negative Controls in Upper Let w How many replicates do you need on How many negative controls do you need for each target assay 3 For each sample in the reaction plate enter a sample name and select a sample popi popi popi pop2 pop2 pop2 IN Gi N G N bi mF m g m m m S
96. ftware analyzes the data using the standard curve quantitation method Section 1 of this chapter explains how to review the analyzed data using several of the analysis screens and how to publish the data If you obtain questionable results Section 2 of this chapter explains how to perform some troubleshooting steps Example Start Experiment Experiment WV Workflow Design the Experiment Chapter 2 WwW Prepare the Reactions Chapter 3 v Run the Experiment Chapter 4 WwW Analyze the Experiment Chapter 5 Section 1 Review Results Analyze View the standard curve View the amplification plot View the results in a table Publish the data Section 2 Troubleshoot If Needed 1 View the analysis settings adjust the baseline threshold 0O A OO N 2 View the quality summary 3 Omit wells 4 View the multicomponent plot 5 View the raw data plot End Experiment Notes 66 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment Section 5 1 Review Results Section 5 1 Review Results This section covers Amro ERA Ts SERES dE a SS ee er ane a rere 68 E View the Standard Curve sscrsdssosgaadad ds bs Ed DD CAES PU ee ees 71 E View the Amplification Plot onana 000 ccc nes 74 E View the Well Table s 4544s4444 4455044 da Ad ee deb ood eed eeu es 81 DOME OA i oe oc hee n e 6 oe
97. i Experiments Preface Safety Conventions Used in This Document Safety Conventions Used in This Document Safety Alert Words xii Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical AN o7 Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices Nem Indicates a potentially hazardous situation that if not avoided could result in death or serious injury AN DUYN Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard icons that are affixed to Applied Biosystems instruments see Safety Symbols on page xiii Examples The following examples show the use of safety alert words IMPORTANT Wear powder free gloves when you han
98. ication of the internal positive control IPC In the 7500 7500 Fast system software the task for the IPC target in wells that contain IPC blocking agent See also negative control blocked IPC wells See threshold cycle CT See reference sample See reagents Method for determining relative target quantity in samples With the comparative C AAC method the 7500 7500 Fast system software measures amplification of the target and of the endogenous control in samples and in a reference sample Measurements are normalized using the endogenous control The software determines the relative quantity of target in each sample by comparing normalized target quantity in each sample to normalized target quantity in the reference sample Dye that is not supplied by Applied Biosystems Custom dyes may be adapted for use in experiments on the 7500 7500 Fast system When using custom dyes the custom dye should be added to the Dye Library and a custom dye calibration performed See threshold cycle CT In the thermal profile a stage that is repeated A cycling stage is also called an amplification stage For cycling stages you can enable AutoDelta settings See also amplification stage Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 111 Experiments Glossary data collection delta Rn ARn derivative reporter Rn Design Wizard diluent Diluted Sample Concentration 10X for Reaction
99. ick the arrow beneath the Design Wizard icon to expand the Set Up menu d Create a new experiment or open an existing experiment e Select File Import f Click Browse locate and select the text file txt then click Select g Click Start Import The setup data from the exported text file is imported into the open experiment Note If your experiment already contains plate setup information the software prompts you to replace the plate setup with the data from the text file Click Yes to replace the plate setup 2 Use Advanced Setup to finish setting up your experiment see page 98 3 Prepare the PCR reactions Experiment Type Prepare the Relative standard curve Template Sample dilutions Standard dilution series Reaction mix Reaction plate Standard curve o Ooo ob Comparative Cr a Template Genowpng b Pepe enon c Reaction mix Presence absence d Reaction plate 4 Run the experiment IMPORTANT While the 7500 7500 Fast instrument is performing a run do not create experiments perform maintenance or allow the computer to run antivirus software or to enter hibernation Performing such activities while the instrument is running an experiment will cause gaps in data collection Load the reaction plate into the instrument a b Start the run c Optional Monitor the run d Unload the reaction plate from the instrument Notes Applied B
100. iency measurements include replicates to decrease the effects of pipetting inaccuracies PCR inhibitors PCR inhibitors in the reaction can reduce amplification efficiency e R values correlation coefficient The R value is a measure of the closeness of fit between the regression line and the individual C data points of the standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points An R value gt 0 99 is desirable e Cy values The threshold cycle C is the PCR cycle number at which the fluorescence level meets the threshold A Cy value gt 8 and lt 35 is desirable A Cy value lt 8 indicates that there is too much template in the reaction A Cy value gt 35 indicates a low amount of target in the reaction for Cy values gt 35 expect a higher standard deviation If your experiment does not meet the guidelines above troubleshoot as follows e Omit wells see Omit Wells from the Analysis on page 90 Or e Rerun the experiment For More For more information on Information The Standard Curve screen Open the 7500 Software Help by clicking or pressing F1 e Amplification efficiency Refer to the Amplification Efficiency of TaqMan Gene Expression Assays Application Note Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 13 Experiments m Chapter 5 Analyze the Experiment View the A
101. igation pane select Analysis gt Data Plot Note If no data are displayed click Analyze 2 Display all 96 wells in the Raw Data Plot screen by clicking the upper left corner of the plate layout in the View Plate Layout tab 3 Click Show a legend for the plot Note This is a toggle button When the legend is displayed the button changes to Hide the plot legend The legend displays the color code for each row of the reaction plate In the example shown below Row A is red Row B is yellow green Row C is green and so on 4 Click drag the Show Cycle pointer from cycle 1 to cycle 40 In the example experiment there is a stable increase in signal from filter 1 which corresponds to the FAM dye filter Notes 94 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment me View the Raw Data Plot J Raw Data Plot 3 a ig E Raw Data Plot 4 500 000 4 000 000 3 500 000 3 000 000 2 500 000 B o 2 000 000 1 500 000 1 000 000 500 000 o 1 2 3 4 5 Filter Legend HA De Mic Mo We Mr Mic WH Show Cycle 40 4 The filters are Filter 1 2 3 4 5 e FAM dye e JOE dye TAMRA dye e ROX dye Cy5 dye Dye e SYBR Green VIC dye e NED dye e Texas Red dye e Cy3 dye dye Analysis When you analyze your own st
102. in the PCR reactions and use a specific buffer Review the standard dilution series calculations for each target If needed edit the Standard Concentration in Stock including units Note For the Standard Concentration in Stock units field you can select ng or ug in the drop down list or you can enter another unit in the field for example copies IU International Units nmol pg and so on The table is updated according to your entry Review the sample dilution calculations for each sample If needed edit the diluted sample concentration including units and stock concentration For More For more information on Information Notes Completing the Reaction Setup screen Open the 7500 Software Help by clicking or pressing Fl Applied Biosystems assays Refer to the TagMan Gene Expression Assays Protocol Custom TagMan Gene Expression Assays Protocol 36 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment Order Materials for the Experiment Order Materials for the Experiment In the Materials List screen review the list of materials recommended to prepare the PCR reaction plate Optional Print the materials list create a shopping list then order the recommended materials from the Applied Biosystems Store Note To open the Applied Biosystems Store you need to have an Internet
103. in the plate layout then click Analyze Navigation Tips How to Select Wells To display specific wells in the analysis screens select the wells in the View Plate Layout tab as follows 1 To select wells of a specific type using the Select Wells With drop down lists select Sample Target or Task then select the sample target or task name 2 To select a single well click the well in the plate layout 3 To select multiple wells click drag over the desired wells or CTRL click or Shift click the desired wells in the plate layout 4 To select all 96 wells click the upper left corner of the plate layout gt View Plate Layout View Well Table 1 Select Wells With Select Item O ShowinWells E View Legend 4 sauna a E ne ines SH Ge SO JEn tr Eon BRDN RELS 80 KE 26 3 Saa fm Wells 1 6 Unknown EJ 15 Standard 1 3 Negative Control 72 Empty Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 69 Experiments no Chapter 5 Analyze the Experiment nd Analyze the Experiment How To Display Multiple Plots Use the Multiple Plots screen to display up to four plots simultaneously To navigate within the Multiple Plots screen 1 In the navigation pane select Analysis gt A Multiple Plots View 2 To display four plots click 45 Show plots in a 2 X 2 matrix 3 To display two plots in rows click Show plots in t
104. ing View Plate Layout Notes View Well Table Select Wells With Select Item Well Omit Flag RNase P TAMRA NTC 1 Al 2 A2 3 A3 RNase P TAMRA STANDARD 4 A10 5 A11 6 A12 O RNase P TAMRA STANDARD 7 B7 8 B8 9 B9 RNase P TAMRA STANDARD 10 B4 11 B5 12 B6 d RNase P TAMRA STANDARD Sample 10000 0 1250 0 2500 0 5000 0 Target N RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT Task NTC NTC NTC STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD Dyes FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA CT Undetermi Undetermi Undetermi 26 389278 26 213354 26 242676 29 64913 29 546368 29 421392 28 325405 28 28138 28 69632 Cr Mean 26 281769 26 281769 26 281769 29 538963 29 538963 29 538963 28 434366 28 434366 28 434366 Cr SD 0 094 0 094 0 094 0 114 0 114 0 114 0 228 0 228 0 228 Quantity 10 000 10 000 10 000 1 250 1 250 1 250 2 500 2 500 2 500 Quantity Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments 81 a Chapter 5 Anal
105. iosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 105 Experiments A Notes Appendix A Alternate Exper Export Import Workflow iment Workflows 5 Analyze the data a b Open the experiment in the 7500 software In the navigation pane click Analysis If the data are not analyzed click Analyze In the navigation pane select an analysis screen to view the data for example select QC Summary to view a quality summary of the data 106 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Bibliography Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 107 Experiments Bibliography 108 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Advanced Setup AIF allele allelic discrimination plot amplicon amplification amplification efficiency EFF amplification plot Glossary In the 7500 7500 Fast system software a feature that allows you to set up your experiment according to your experiment design Advanced Setup
106. l 27763 20483 28183 53183 5583 5543 JA ME El RNase El RNase El RNase El RNase El Riase El RNase El Riase El RNase El RNase EI RNase El Riase S RNase 5E3 SEM 5E3 2 SER 2 SER 2 SER 1 25 1 25E3 1 25E3 625 625 625 Wells 1 6 Unknown 5 15 Standard 9 3 Negative Control 72 Empty About the In this getting started guide you will use two files l Example e In Chapter 2 you will create a standard curve example experiment that contains Experiment Data setup data then you will save the file to your computer e In Chapter 5 you will view results in a standard curve example experiment file that contains run data The data file for the example experiment is installed with the 7500 software You can find the data file for the example experiment on your computer at lt drive gt Applied Biosystems lt software name gt experiments Standard Curve Example eds where lt drive gt is the computer hard drive on which the 7500 software is installed lt software name gt is the current version of the 7500 software Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 13 Experiments Chapter 1 Get Started Example Experiment Workflow Example Files in the Experiments Folder The experiments folder of the 7500 software contains several example files that you can reference when analyzing your own data The following example files install with the 7
107. l Instrument Safety AINETE PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Moving and Ave PHYSICAL INJURY HAZARD The instrument 1s to be moved Lifting the and positioned only by the personnel or vendor specified in the applicable site Instrument preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons Moving and Nee Do not attempt to lift or move the computer or the monitor without Lifting Stand the assistance of others Depending on the weight of the computer and or the monitor Alone Computers moving them may require two or more people and Monitors 722 HJHS H J7HS SHYYYS _t _WT_T nn a a a a a a aeaee Things to consider before lifting the computer and or the monitor e Make sure that you have a secure comfortable grip on the computer or the monitor when lifting e Make sure that the path from where the object is to where it is being moved is clear of obstructions e Do not lift an object and twist your torso at the same time Ke
108. leshooting adjust baseline 87 adjust threshold 87 flags 89 omit wells 90 view analysis settings 86 view Multicomponent Plot screen 92 view QC Summary screen 88 view Raw Data Plot screen 94 U unload instrument 64 user attention words described viii using this guide as a tutorial 11 with your own experiments 11 W WARNING description xii waste disposal guidelines xix Well Table 81 wells negative control 28 41 51 omit 90 selecting 69 standard 28 41 51 unknown 28 41 51 workflows Advanced Setup 98 example experiment 14 Export Import 11 104 QuickStart 11 100 126 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments www appliedbiosystems com Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applied Biosystems is committed to providing the world s leading technology and information for life scientists Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 06 2010 Applied Biosystems Part Number 4387779 Rev C
109. leted To notify recipients by email when the instrument completes a run e Obtain e mail addresses to receive notifications IMPORTANT Separate addresses with a comma e Contact your systems administrator or information technology department if you need E mail addresses for users who will receive notifications A network address for a simple mail transfer protocol SMTP server on the LAN A user name and password for the server if required for access The Secure Sockets Layer SSL setting of the server on or off Notes 60 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 4 Run the Experiment no Start the Run Start the Run IMPORTANT While the 7500 7500 Fast instrument 1s performing a run do not create experiments perform maintenance or allow the computer to run antivirus software or to enter hibernation mode Performing such activities while the instrument 1s running an experiment will cause gaps in data collection To start your 7500 7500 Fast instrument 1 Inthe 7500 software click ME Run in the navigation pane 2 Click START RUN p Estimated Time Remaining 56 min 22 sec Instrument Status JA Connected wg lification Plot Run Status L O Enable Notifications mpification Current Temperatures Temperature Plot Wi cover Wi sample Notification Settings Temperature Plot Block
110. lied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve iil Experiments FINISH the DESIGN WIZAIG spas bos pas DES de DEE dr bod eed dete oe oben 40 Chapter3 Prepare the Reactions 00 eens 43 Chapter ONCE ssi acdc ie E eat hk bee ala Cm ee eee wae ees 44 Prepare the Sample Dilutions 0 naaa ee ees 45 Prepare the Standard Dilution Series 0 0 0 es 47 Prepare the Reaction Mix 0 0 0 ee ees 49 Prepare the Reaction Plate 0 0 0 cc eas 51 Chapter 4 Run the Experiment 0 0 0 cee ee 55 Chapter OVeCVIEW pias den DEE Eea TUE DRE E ra gt een 56 Prepare tor he RUM sas pre as aa RG ee DA Sad SRA Pes 57 Enable the Notification Settings Optional 0 0 0 0 ccc eee 59 OVA Gill eM aoe dd E E ae E Be aati eae tego Ny inne ede arte yen a 61 MONTONE RUM Soiree ie teeta ee ee na SEA BEES te ahi Ge ants Sle otek ie ee na ates 61 WNIOAG ANE INSIFUMENE a aus veut a ect be eam eM i es Sa O a aah Ree al Se oe erga 64 Chapter5 Analyze the Experiment 0 0 00 e eee eaes 65 Chapier OVERVIEW ups nda ac A ee Ath we Me Batata en Gee DA be Gul aa 66 Sections 1 Review ReSUlls sussa tedster aa beeen teas 67 Analyze the Experiment 0 0000 eee eee eee 68 View the Standard Curve cc ee eee ete eee 71 View the Amplification Plot 0 0 ccc eee eee 74 VIEW tne Well TADS ress aod weed at ee qe se E a oe A RAS ee A 81 PUDNSH NE Wal at xe a
111. ll If you want to set up a standard curve experiment with multiplex reactions amplification and detection of two or more targets per well design your experiment using Advanced Setup see page 98 instead of the Design Wizard Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 29 Experiments Chapter 2 Design the Standard Curve Experiment Set Up the Run Method Set Up Biological Replicate Groups 1 Select Specify Biological Replicate Groups 2 Enter the number of biological replicate groups that you want to test in the reaction plate 3 For each biological replicate group click the cell in the Biological Group Name column then enter a name for the biological group For example BrainGP 4 Assign the biological replicate group s to the reaction plate a Select wells in the reaction plate that contain samples associated with the biological replicate group b In the Assign column select the check box of the appropriate biological replicate group Set Up Biological Replicate Groups No Biological Replicates Specify Biological Replicate Groups 1 How many Biological Replicate Groups do you want to test in the reaction plate o 2 Select well s in the plate layout then assign a biological replicate group from the list Assign Biological Group Name Color Comments 4b prince Ml For More For more information on Information e Completing the Samples screen O
112. lls 72 Empty wells e The plate layout that the 7500 software automatically generates is used Review Plate Layout for Experiment Standard Curve Example 3 Review the plate layout then select what you wantto do next Save Experiment Start Run for This Experiment Edit Plate Layout phy bala orl Return to the Wizard Save and close this Save this experiment then Use Advanced Setup to edit Save and close this Continue designing this experiment start the run Make sure the the plate layout experiment then create experiment using the Design reaction plate is loaded into another experiment using the Wizard the instrument Design Wizard fy ee g p ye EDS ES a Ey ieee pae ga p tee a Ey Pee ES gase ae ES eee Ey eee Ey ee Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 51 Experiments NQ Chapter 3 Prepare the Reactions Prepare the Reaction Plate Required e Microcentrifuge tubes Materials e Pipettors e Pipette tips e RNase P reaction mix from page 49 e Water e Standards from page 47 Samples from page 45 e MicroAmp Optical 96 Well Reaction Plate e MicroAmp Optical Adhesive Film e Centrifuge Prepare the 1 Prepare the negative control reactions for the target Reaction Plate a To an appropriately sized tube add the volumes of reaction mix and water listed below Tube Reaction Mix Reaction Mix Volume uL Water Volume uL
113. lluminates all wells of the reaction plate within the instrument exciting the fluorophores in each reaction 2 Emission The instrument optics collect the residual fluorescence emitted from the wells of the reaction plate The resulting image collected by the device consists only of light that corresponds to the range of emission wavelengths 3 Collection The instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval The 7500 software stores the raw fluorescence image for analysis After a run the 7500 software uses region of interest ROI optical dye and background calibrations to determine the location and intensity of the fluorescence in each read the dye associated with each fluorescent signal and the significance of the signals Notes 2 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 1 Get Started 4 About the 7500 7500 Fast System About the Filters The 7500 7500 Fast system uses the following filters Filter 1 2 3 4 5 e FAM dye e JOE dye TAMRA dye e ROX dye Cy5 dye Dye e SYBR Green e VIC dye NED dye e Texas Red dye e Cy3 dye dye For More For information on Information The 7500 7500 Fast system Refer to the Applied Biosystems 7500 7500 Fast Real Time PCR Software Help Note To open the Help select Help
114. ly S ly S i ara ara ara color Enter Sample Name gt pop1 a gr gr Hg ag SE gr a Hg 8 Ss gF ac ac crs cera er bh oe I Bee el ol ts ot CM 4 anc enc enc enc 01 Design When you design your own standard curve experiment Guidelines Notes Identify each sample with a unique name and color You can enter up to 100 characters in the Sample Name field Enter the number of replicates identical reactions to set up Applied Biosystems recommends three replicates for each sample reaction Enter the number of negative control reactions to set up Applied Biosystems recommends three negative control reactions for each target assay You can set up biological replicate groups see Set Up Biological Replicate Groups Biological replicates allow you to assess the representative nature of your results as they relate to the population being studied Inclusion of biological replicates can give insight into any natural variation that is present within the population Select which targets to test in the samples Select All Sample Target Reactions to test all targets in all samples Select Specify Sample Target Reactions to specify the targets to test in each sample IMPORTANT When you use the Design Wizard to set up a standard curve experiment you can set up only singleplex reactions amplification and detection of one target per we
115. master mix added to the reaction If you use SYBR Green dye select the Include Melt Curve check box to perform melt curve analysis of the amplified target Note Although you can use other fluorescence based reagents on the 7500 7500 Fast system you must design your experiment using Advanced Setup instead of the Design Wizard e Select the appropriate ramp speed for the instrument run Select Fast 40 minutes to complete a run if you are using Fast reagents for the PCR reactions Select Standard 2 hours to complete a run if you are using standard reagents for the PCR reactions e Select the appropriate PCR template Select cDNA complementary DNA if you are performing 2 step RT PCR and you have already performed reverse transcription to convert the RNA to cDNA You are adding complementary DNA to the PCR reactions Select RNA if you are performing 1 step RT PCR You are adding total RNA or mRNA to the PCR reactions Note To use the Fast ramp speed with RNA templates you must design your experiment using Advanced Setup instead of the Design Wizard Select gDNA genomic DNA if you have already extracted the gDNA from tissue or sample You are adding purified genomic DNA to the PCR reactions Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 23 Experiments O Chapter 2 Design the Standard Curve Experiment Set Up the Targets For More For mor
116. me Total Volume Standard Concentration Point HL HL HL ng L 1 10 000 Stock 3 63 14 52 18 15 4000 0 2 5 000 Dilution 1 9 08 9 08 18 15 2000 0 3 2 500 Dilution 2 9 08 9 08 18 15 1000 0 4 1 250 Dilution 3 9 08 9 08 18 15 500 0 5 625 Dilution 4 9 08 9 08 18 15 250 0 Required e Water to dilute the standards Materials e Microcentrifuge tubes e Pipettors e Pipette tips e Standard stock e Vortexer e Centrifuge Prepare the 1 Label a separate microcentrifuge tube for each standard Standard Dilution e RNase P Std 1 Series for the e RNase P Std 2 RNase P Assay a e RNase P Std 3 e RNase P Std 4 e RNase P Std 5 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 47 Experiments 2 Chapter 3 Prepare the Reactions Prepare the Standard Dilution Series 2 Add the required volume of water diluent to each empty tube Tube Standard Name Volume of Diluent to Add uL 1 RNase P Std 1 9 08 2 RNase P Std 2 9 08 3 RNase P Std 3 9 08 4 RNase P Std 4 9 08 5 RNase P Std 5 9 08 3 Prepare dilution 1 in the RNase P Std 1 tube a Vortex the stock for 3 to 5 seconds then centrifuge the tube briefly b Using a new pipette tip add 9 08 uL of stock to the RNase P Std 1 tube c Vortex Std 1 for 3 to 5 seconds then centrifuge the tube briefly 4 Prepare dilution 2 in the RNase P Std 2 tube
117. moving and lifting instrument xvi moving parts xx moving lifting xvi physical hazard xx repetitive motion xxi standards xxii workstation xxi safety labels on instruments xiv safety standards xxii safety symbols on instruments xiii sample dilutions calculated volumes 34 45 prepare 45 Sample screen 28 samples design guidelines 29 dilutions 45 sample reactions unknowns 53 setup 28 select wells 69 singleplex PCR 8 24 SPIKE flag 89 standard curve experiments about 7 standard deviation effect of threshold on 79 standard dilution series calculated volumes 34 component of experiment 7 prepare 47 standard vs fast plates 6 standards component of experiment 7 design guidelines 27 diluting 47 enable notification settings 59 EMC xxii for more information 63 safety xxii guidelines 60 setup 26 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 125 Experiments Index Set Up Standards check box 24 25 Template 11 102 standard reactions 52 workstation safety xxi Standards screen 26 SYBR Green reagents 3 9 23 24 25 36 95 symbols safety xiii T TaqMan reagents 3 9 23 24 25 36 95 targets design guidelines 25 setup 24 Targets screen 24 Technical Support contacting xi Template 11 102 template See samples text conventions viii THOLDFAIL flag 89 threshold correct values 78 examples 79 manually adjust 87 training information on xi troub
118. mplification Plot View the Amplification Plot The Amplification Plot screen displays amplification of all samples in the selected wells Three plots are available e ARn vs Cycle ARn is the magnitude of normalized fluorescence generated by the reporter at each cycle during the PCR amplification This plot displays ARn as a function of cycle number You can use this plot to identify and examine irregular amplification and to view threshold and baseline values for the run e Rn vs Cycle Rn is the fluorescence from the reporter dye normalized to the fluorescence from the passive reference This plot displays Rn as a function of cycle number You can use this plot to identify and examine irregular amplification e Cr vs Well C is the PCR cycle number at which the fluorescence meets the threshold in the amplification plot This plot displays Cy as a function of well position You can use this plot to locate outlying amplification outliers Each plot can be displayed on a linear or log10 scale About the In the standard curve example experiment you review the target in the Amplification Example Plot screen for Experiment e Correct baseline and threshold values e Outliers View the 1 Inthe navigation pane select Analysis gt Amplification Plot Amplification Plot Note If no data are displayed click Analyze 2 Display the RNase P wells in the Amplification Plot screen a Select the View Plate Layout tab
119. n d Unload the reaction plate from the instrument Notes 100 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Appendix A Alternate Experiment Workflows QuickStart Workflow 4 In the 7500 software complete the plate setup Experiment Type Select and complete the Genotyping a Define SNP Assays and Samples tab b Assign SNP Assays and Samples tab All other experiments a Define Targets and Samples tab b Assign Targets and Samples tab 5 Analyze the data Q Open the experiment in the 7500 software b In the navigation pane click Analysis c If the data are not analyzed click Analyze d In the navigation pane select an analysis screen to view the data for example select QC Summary to view a quality summary of the data Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 101 Experiments A Appendix A Alternate Experiment Workflows Template Workflow Template Workflow Create a Template Create an Experiment with a Template Notes 1 You can use a template to create a new experiment Templates allow you to create many experiments with the same setup information Double click 3 7500 software or select Start gt All Programs gt Applied Biosystems gt 7500 Software gt lt software name gt where lt software name gt is the current version
120. n efficiency values are affected by the C of well B12 which you will omit later in this guide Standard Curve Note If no data are displayed click Analyze Display all 96 wells in the Standard Curve screen by clicking the upper left corner of the plate layout in the View Plate Layout tab Inthe Target drop down list select All In the Plot Color drop down list select Default Click Show a legend for the plot Note This is a toggle button When the legend is displayed the button changes to Hide the plot legend View the values displayed below the standard curve In the example experiment the values for the target RNase P are within the acceptable ranges The slope is 3 685 The R value is 0 992 The amplification efficiency Eff is 86 81 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 11 Experiments Chapter 5 Analyze the Experiment View the Standard Curve f Check that all samples are within the standard curve In the example experiment all samples blue dots are within the standard curve red dots Standard Curve Plot Settings Save current settings as the default 4 _ a hets Standard Curve 31 0 m 30 5 30 0 29 5 29 0 5 285 7 28 0 27 5 27 0 26 5 26 0 Quantity 6 Target RNase P TAMRA Slope 3 685 Y Inter 40 966 p 0 992 Eff 86 81 Legend E Standa
121. n mix using the components and volumes that were calculated by the 7500 software Complete the Reaction Mix Calculations Tab see page 33 on page 32 Note The software calculates the volumes for all components for the PCR reactions However when you prepare the reaction mix in this section include only the master mix assay mix and water Add the sample or standard when you prepare the reaction plate see Prepare the Reaction Plate on page 51 About the For the standard curve example experiment Example The reaction mix components are EXPERIMENT TaqMan Universal PCR Master Mix 2X or TaqMan 2X Universal PCR Master Mix No AmpErase UNG RNase P Assay Mix 20X Water e The volumes calculated in the software are Component Volume for 1 Reaction pL Master Mix 2 0X 12 50 Assay Mix 20 0 1 25 H O 8 75 Total Volume 22 50 Note The sample or standard is not added at this time Required e Microcentrifuge tubes Materials Pipettors e Pipette tips e Reaction mix components listed above e Centrifuge Prepare the N ON CHEMICAL HAZARD TaqMan Universal PCR Master Mix 2X Reaction Mix may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting
122. nd data storage file transfer and copying and pasting Have networking experience if you plan to integrate the 7500 7500 Fast system into your existing laboratory data flow Text Conventions This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File Open User Attention Two user attention words appear in Applied Biosystems user documentation Each word Words implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate reagent kit use or safe use of a chemical Examples of the user attention words appear below Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection you need a valid user ID viii Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface How to Obtain More Information Safety Alert Safety alert words also appear in
123. ndard ramp speed is used in the instrument run extract the gDNA from tissue or sample Complete the 1 Select Standard Curve as the quantitation method Methods amp Materials Screen Select TaqMan Reagents for the reagents Select gDNA genomic DNA for the template type o 2 Pb Click Next 1B Define Methods amp Materials Q Instructions Selectthe quantitation method reagents ramp speed and type of template for the real time PCR reactions Which quantitation method are you using With the standard curve method you use standards to determine the absolute quantity of target sequence in a sample Which reagents do you want to use to detect the target sequence Purified gDNA isolated from two populations is the template type You must first Select Standard 2 hours to complete a run for the ramp speed Methods amp Materials Help These real time PCR reactions contain two primers and a TaqMan probe The primers are designed to amplify the target sequence The TaqMan probe is designed to hybridize to the target sequence and generate fluorescence signal when the target sequence is amplified Which ramp speed do you want to include in the instrument run 3 3 Standard 7 nous to complete un For optimal results using the standard ramp speed Applied Biosystems recommends standard reagents for your real time PCR reactions What type of template do you want to use in the real time PCR reactions
124. ndards c UL us Canadian EMC Standard European Safety and EMC Standards Ce Australian EMC Standards Xxii This section provides information on e U S and Canadian Safety Standards e Canadian EMC Standard e European Safety and EMC Standards e Australian EMC Standards This instrument has been tested to and complies with standard UL 61010A 1 Safety Requirements for Electrical Equipment for Laboratory Use Part 1 General Requirements and with standard UL 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials This instrument has been tested to and complies with standard CSA 1010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements This instrument has been tested to and complies with ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators Safety This instrument meets European requirements for safety Low Voltage Directive 2006 95 EC This instrument has been tested to and complies with standards EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements and EN 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials and with standard EN 61010 2 081 2002 A1 2003 Particular Requirements for Automatic and Semi Automatic Laboratory Equipment fo
125. nes When you design your own standard curve experiment If you are using TaqMan reagents select the type of assay you are using Select Inventoried Made to Order if you are using Applied Biosystems TaqMan Gene Expression Assays Inventoried or Made to Order or Applied Biosystems Custom TaqMan Gene Expression Assays Select Custom if you are designing your own assays with Primer Express software Enter a reaction volume well Applied Biosystems recommends a reaction volume of 25 uL for standard curve experiments The 7500 system supports reaction volumes from 20 to 100 uL The 7500 Fast system supports reaction volumes from 10 to 30 uL Include excess reaction volume to account for the loss that occurs during pipetting Applied Biosystems recommends an excess reaction volume of at least 10 Review the reaction mix concentrations for each target If needed For TaqMan reagents edit the master mix and assay mix concentrations For SYBR Green reagents edit the master mix forward primer and reverse primer concentrations For 1 step RT PCR edit the reverse transcriptase concentration Review the reaction mix components for each target If you are running Fast PCR reactions make sure you use Fast master mix in the PCR reactions If you are running standard PCR reactions make sure you use standard master mix in the PCR reactions For l step RT PCR make sure you include reverse transcriptase
126. nical replicates or biological replicates Fluorescent dye used to detect amplification If you are using TagMan reagents the reporter dye is attached to the 5 end If you are using SYBR Green reagents the reporter dye is SYBR Green dye An oligonucleotide that flanks the 3 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target An enzyme that converts RNA to cDNA Reverse transcriptase is added to the PCR reaction to perform 1 step RT PCR See normalized reporter Rn A dye supplied by Applied Biosystems and precalibrated on the 7500 7500 Fast systems ROX dye is used as the passive reference See refSNP ID Definition of the reaction volume and the thermal profile for the 7500 7500 Fast instrument run The template that you are testing In the 7500 7500 Fast system software a reaction component displayed on the Reaction Mix Calculations tab of the Reaction Setup screen The software assumes the sample DNA is added to the reaction mix at a 10X concentration For example if the reaction volume is 20 uL the calculated volume of sample for 1 reaction is 2 uL Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 117 Experiments Glossary Sample Library Sample or Standard 10x sample SNP assay reaction sample target reaction serial factor series slope SNP SNP assay SNP Assay Lib
127. nly the IPC template should amplify in negative control IPC wells because the reaction contains no sample Previously called IPC See negative control blocked IPC wells See negative control NC Molecules that are attached to the 3 end of TaqMan probes When the probe is intact the nonfluorescent quencher NFQ prevents the reporter dye from emitting fluorescence Because the NFQ does not fluoresce it produces lower background signals resulting in improved precision in quantitation The minor groove binder MGB increases the melting temperature Tm without increasing probe length It also allows the design of shorter probes Quantity of target divided by the quantity of endogenous control Fluorescence from the reporter dye normalized to the fluorescence of the passive reference An action that you perform before reanalysis to omit one or more wells from analysis Because no algorithms are applied to omitted wells omitted wells contain no results For a set of data a datapoint that is significantly smaller or larger than the others Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments passive reference plate layout point positive control post PCR read pre PCR read primer mix primer probe mix pure dye quantitation method quantity quencher Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve
128. nmental Symbols on Instruments The following symbol applies to all Applied Biosystems electrical and electronic products placed on the European market after August 13 2005 Symbol Description Do not dispose of this product as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to reduce the EE environmental impact of waste electrical and electronic equipment WEEE A l European Union customers Call your local Applied Biosystems Customer Service office for equipment Lo pick up and recycling See http www appliedbiosystems com for a list of customer service offices in the European Union E Safety Labels on Instruments The following CAUTION WARNING and DANGER statements may be displayed on Applied Biosystems instruments in combination with the safety symbols described in the preceding section English CAUTION Hazardous chemicals Read the Material Safety Data Sheets MSDSs before handling Francais ATTENTION Produits chimiques dangeureux Lire les fiches techniques de s ret de mat riels avant la manipulation des produits CAUTION Hazardous waste Refer to MSDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de s ret de mat riels et la r gulation locale associ es la manipulation et l limination des d chets WARNING Hot lamp AVERTISSEMENT Lampe br lante WARNI
129. ns Part of the run method that specifies the temperature time ramp and data collection points for all steps and stages of the 7500 7500 Fast instrument run 1 In amplification plots the level of fluorescence above the baseline and within the exponential growth region The threshold can be determined automatically see automatic CT or can be set manually see manual CT 2 In presence absence experiments the level of fluorescence above which the 7500 7500 Fast system software assigns a presence call The PCR cycle number at which the fluorescence meets the threshold in the amplification plot See melting temperature Tm In the 7500 7500 Fast system software the task for the target or SNP assay in wells that contain the sample you are testing e In quantitation experiments the task for the target in wells that contain a sample with unknown target quantities In genotyping experiments the task for the SNP assay in wells that contam a sample with an unknown genotype In presence absence experiments the task for the target in wells that contain a sample in which the presence of the target is not known In presence absence experiments wells that contain a sample and internal positive control IPC y intercept In the standard curve the value of y where the regression line crosses the y axis The y intercept indicates the expected threshold cycle C for a sample with quantity equal to 1 Applied Biosystems 7500 7500
130. nt IMPORTANT While the 7500 7500 Fast instrument is performing a run do not create experiments perform maintenance or allow the computer to run antivirus software or to enter hibernation Performing such activities while the instrument is running an experiment will cause gaps in data collection Load the reaction plate into the instrument a b Start the run O Optional Monitor the run Q Unload the reaction plate from the instrument f Analyze the data Q Open the experiment in the 7500 software oO In the navigation pane click Analysis c If the data are not analyzed click Analyze d In the navigation pane select an analysis screen to view the data for example select QC Summary to view a quality summary of the data Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 103 Experiments A Appendix A Alternate Experiment Workflows Export Import Workflow Export Import Workflow Use the Export Import workflow to set up a new experiment using setup data exported from another experiment Only reaction plate setup data are exported and imported Export Setup 1 Double click 3 7500 software or select Start gt All Programs gt Data Applied Biosystems gt 7500 Software gt lt software name gt where lt software name gt is the current version of the 7500 software 2 Open an existing experiment or create a new experiment
131. nt the Frequency column displays for the OUTLIERRG flag Note A 0 that is displayed in the Frequency column indicates that the flag does not appear in the experiment 4 Optional Click the OUTLIERRG flag row to display details about the flag Flag Summary Total Wells 96 Processed Wells 24 Manually Omitted Wells 0 Targets Used Wells Set Up 24 Flagged Wells 1 Analysis Omitted Wells O Samples Used Flag Details Name Tepee 4 Amplification in negative control 0 BADROX Bad passive reference signal O Ud OFFSCALE Fluorescence is offscale O o HIGHSD High standard deviation in replicate group D UN NOAMP No amplification es NOISE Noise higher than others in plate O o QO SPIKE Noise spikes NOSIGNAL No signal in well a OUTLIERRG Outlier in replicate group a SL EXPFAIL Exponential algorithm failed OD dA BLFAIL Baseline algorithm failed OD dA THOLDFAIL Thresholding algorithm failed Do CTFAIL Cr algorithm failed OD dA EEE Flag OUTLIERRG Outlier in replicate group Flag Detail The CT deviates significantly from CT values in the associated replicate group Flagged Wells B12 View OUTLIERRG Troubleshooting Information Notes 88 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Chapter 5 Analyze the Experiment View the QC Summary Possible Flags For standard curve experiments the flags listed below can be gene
132. nt using a 4 or 5 color 96 well system 01 What type of experiment do you want to design Design a gene quantitation experiment to determine the amount of target nucleic acid sequence in a sample Notes 20 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Design Guidelines Chapter 2 Design the Standard Curve Experiment o Define the Experiment Properties When you design your own standard curve experiment you e Enter an experiment name that is descriptive and easy to remember You can enter up to 100 characters in the Experiment Name field You cannot use the following characters in the Experiment Name field gt lt Note The experiment name 1s used as the default file name Optional Enter a barcode to identify the barcode on the reaction plate You can enter up to 100 characters in the Barcode field Optional Enter a user name to identify the owner of the experiment You can enter up to 100 characters in the User Name field Optional Enter comments to describe the experiment You can enter up to 1000 characters in the Comments field Select the instrument you are using to run the experiment 7500 96 Wells 7500 Fast 96 Wells Note You can use 7500 software v2 0 to design experiments for the 7500 7500 Fast instrument Note To set the default instrument type select Tools Preferences then select the
133. oAmp Optical 8 Cap Strip D E MicroAmp Optical Adhesive Film Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments ao Chapter 1 Get Started Supported Consumables Standard vs Fast Reaction Plates and Tubes Notes Make sure that you use the correct reaction plate for your system System Reaction Plate 7500 system e MicroAmp Optical 96 Well Reaction Plate PN 4306737 also called standard reaction plates e MicroAmp Optical Tubes without Caps 0 2 mL PN N8010933 also called standard reaction tubes e MicroAmp Reaction Tubes with Caps 0 2 mL PN N2070540 e MicroAmp Optical 8 Tube Strip 0 2 mL PN 4316567 also called standard reaction tube strips IMPORTANT Fast reaction plates and tubes do not fit into the standard sample block correctly and will result in loss of data 7500 Fast e MicroAmp Fast Optical 96 Well Reaction Plate PN 4346906 also called system Fast reaction plates e MicroAmp Fast 8 Tube Strip 0 1 mL PN 4358293 also called Fast reaction tube strips IMPORTANT Standard reaction plates and tube strips will not properly function and might be crushed when using the Fast sample block Fast Reaction Plates Standard Reaction Plates PN 4346906 PN 4306737 E Notch at top right E corner by A12 T Notch at a gmail top left atu 4 corner TE y U u by A1 E fi T l T
134. obe labeled with VIC dye detects the endogenous control IMPORTANT SYBR Green reagents cannot be used for multiplex PCR DO E Target Primer Set OO Endogenous Control m O L W F ae Primer Set l cDNA Singleplex PCR Multiplex PCR 1 vs 2 Step RT PCR You can perform reverse transcription RT and PCR in a single reaction 1 step or in separate reactions 2 step The reagent configuration you use depends on whether you are performing 1 or 2 step RT PCR e In I step RT PCR RT and PCR take place in one buffer system which provides the convenience of a single tube preparation for RT and PCR amplification However you cannot use Fast PCR master mix or the carryover prevention enzyme AmpErase UNG uracil N glycosylase to perform 1 step RT PCR e 2 step RT PCR is performed in two separate reactions First total RNA is reverse transcribed into cDNA then the cDNA is amplified by PCR This method is useful for detecting multiple transcripts from a single cDNA template or for storing cDNA aliquots for later use The AmpErase UNG enzyme can be used to prevent carryover contamination Note For more information on AmpErase UNG refer to the Applied Biosystems Real Time PCR Systems Reagent Guide Notes 8 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 1 Get Started About Standard Curve Experiments Supp
135. of reaction performed in the well for the target or SNP assay Available tasks e Unknown e Negative Control e Standard standard curve and relative standard curve experiments e Positive control genotyping experiments e IPC presence absence experiments e Blocked IPC presence absence experiments Identical reactions that contain identical components and volumes and evaluate the same sample See also biological replicates Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments template thermal profile threshold threshold cycle C Tm unknown unknown IPC wells Glossary In the Design Wizard of the 7500 7500 Fast system software and in QuickStart for quantitation experiments the type of nucleic acid to add to the PCR reaction The recommended template varies according to experiment type e Quantitation experiments standard curve relative standard curve and comparative Cr cDNA complementary cDNA RNA or gDNA genomic DNA For quantitation experiments the template type selection affects the run method reaction setup and materials list e Genotyping experiments Wet DNA gDNA or cDNA or dry DNA gDNA or cDNA For genotyping experiments the template type selection affects the reaction setup e Presence absence experiments DNA For presence absence experiments Applied Biosystems recommends adding DNA templates to the PCR reactio
136. on Mix Sample Volume Tube Unknown Reaction Reaction Mix Volume uL Sample uL 1 RNase P pop1 RNase P reaction mix 74 25 pop1 8 25 2 RNase P pop2 RNase P reaction mix 74 25 pop2 8 25 b Mix the reactions by gently pipetting up and down then cap the tubes c Centrifuge the tubes briefly to remove air bubbles d Add 25 uL of the unknown sample reaction to the appropriate wells in the reaction plate 4 Seal the reaction plate with optical adhesive film 5 Centrifuge the reaction plate briefly to remove air bubbles 6 Verify that the liquid is at the bottom of each well of the reaction plate If not centrifuge the reaction plate again at a higher speed and for a longer period of time IMPORTANT Do not allow the bottom of the reaction plate to become dirty Fluids and other contaminants that adhere to the bottom of the reaction plate can contaminate the sample block and cause an abnormally high background signal Correct Incorrect ar hoe Liquid is at the e Not centrifuged with enough force or bottom of the well e Not centrifuged for enough time f Place the reaction plate on ice in the dark until you are ready to perform the run Preparation When you prepare your own standard curve experiment Guidelines e Make sure you use the appropriate consumables e Make sure the arrangement of the PCR reactions matches the plate layout displayed in the 7500 software Yo
137. on volume is 25 uL reaction the sample volume is 2 5 uL reaction e The stock concentration is 100 ng uL After diluting the sample according to the Sample Dilutions Calculations table the sample has a concentration of 6 6 ng uL Adding 2 5 uL at this concentration to the final reaction mix volume of 25 uL yields a 1X concentration in the final reaction e The volumes calculated in the software are Sample Stock Concentration Sample Volume Diluent Volume Total Volume of Diluted Sample Name ng pL uL uL uL pop1 100 0 1 0 14 15 15 15 pop2 100 0 1 0 14 15 15 15 Required e Water to dilute the sample Materials i Prepare the 1 Sample Dilutions Microcentrifuge tubes Pipettors Pipette tips Sample stock Vortexer Centrifuge Label a separate microcentrifuge tube for each diluted sample e Population 1 e Population 2 2 Add the required volume of water diluent to each empty tube Tube Sample Name Diluent Volume pL 1 Population 1 14 15 2 Population 2 14 15 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 45 Experiments a Chapter 3 Prepare the Reactions Prepare the Sample Dilutions 3 Add the required volume of sample stock to each tube Tube Sample Name Sample Volume uL 1 Population 1 1 0 2 Population 2 1 0 4 Vortex each diluted sample for 3 to 5 seconds then centrifuge the tubes briefly 5 Place th
138. orted TaqMan and SYBR Green Reagents Reagents Applied Biosystems offers TaqMan and SYBR Green reagents for use on the 7500 7500 Fast system Both reagent types are briefly described in the table below Note If you use TaqMan or SYBR Green reagents the 7500 software automatically calculates reaction volumes in the Reaction Setup screen Reagent Type Process TaqMan reagents or kits PCR and Detection of cDNA a Assay Components Description TaqMan reagents use a fluorogenic probe to enable detection of a specific PCR product as it accumulates during PCR cycles Advantages e Increased specificity with the addition of a fluorogenic probe LEGEND e Provides multiplex capability e a E umas e Preformulated assays optimized to run a Tm W wencher under universal thermal cycling conditions mai icc Moor eroove are available c Signal Generation G AmpliTaq Gold e Can be used for either 1 or 2 step RT PCR DNA Polymerase Limitations o G iii ee Reverse primer a N Requires synthesis of a unique fluorogenic a Ae Fo Mice _ er pro be JOU IIL I S A an pene 3 5 Extended Primer SYBR Green reagents E E SE Ss Step 1 Reaction setup ep to ti tdi AI fluoresces when bound to SYBR Green reagents use SYBR Green dye double stranded DNA a double stranded DNA binding dye to detect PCR products as they accumulat
139. otes 18 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment View the Amplification Plot Threshold Examples Threshold Set Correctly GE The threshold is set in the exponential phase of the amplification curve Threshold settings above or below the optimum increase the standard deviation of the replicate groups n 2 m i 4 Task Dyas CT CT Mean CT SD Quantity Quantity Quantity Gomm HTE FAM HEO Linden HTE FAW HFO Um stemmi HTG FAM ENFO Umasa UMENDIM FAM HFG 20 096757 26 9237796 DOT 2484M TES ATE 1283 UNMMOWN FAM MPO 28 533757 28003756 C074 2887084 2551478 1767 UENO FAM HFG 2006972 20 5923790 DaT 2473004 2551 478 1282 hn k eo 0 007 si as 0 0001 Threshold Set Too Low i The threshold is set below the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Drag the threshold bar up into the exponential phase of the curve 0 14 Task Dyes Er CtMean CrSD Quantity Quantity Quantity E NTC FAMNEO Ungetemi E HTE FAM HFO Umami NTG FAMENFO 38453082 wer UNEM FAMENFO 2285404 27 760 T4de 0252 2314852 2472453 00 435 UMENCWH FAM HFO 22478373 DITA 0 282 2937732 2472483 400 435 UENGA FAM NED 22054218 22 Tdirdd 0 252 2174018 2472403 00
140. p a new experiment with guidance from the software Chapter 2 The Design Wizard guides you through best practices as you create your own experiment The Design Wizard is recommended for new users Note Design options are more limited in the Design Wizard than in Advanced Setup Advanced Set up a new experiment using advanced options page 98 Setup Advanced Setup allows design flexibility as you create your own experiment Advanced Setup is recommended for experienced users QuickStart Run a new experiment with no plate setup information If page 100 desired you can add all design parameters after the run Template Set up a new experiment using setup information from a page 102 template Export Import Import experiment designs from ASCII text files that page 104 contain experiment setup information Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 11 Experiments 4 Chapter 1 Get Started About the Example Standard Curve Experiment About the Example Standard Curve Experiment Notes Description To illustrate how to perform standard curve experiments this guide leads you through the process of designing preparing running and analyzing an example experiment The example experiment represents a typical setup that you can use to quickly familiarize yourself with a 7500 7500 Fast system The objective of the standard curve example experiment is
141. pen the 7500 Software Help by clicking or pressing F1 e Using Advanced Setup See Advanced Setup Workflow on page 98 Set Up the Run Method In the Run Method screen review the reaction volume and the thermal profile for the default run method If needed you can edit the default run method or replace it with one from the Run Method library About the In the standard curve example experiment the default run method is used with one Example change The reaction volume per well is changed from 50 uL to 25 UL Experiment Notes 30 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment Dl Set Up the Run Method Review the Run 1 Select either the Graphical View tab default or Tabular View tab Method Screen Click the Reaction Volume Per Well field then enter 25 uL 3 Make sure the thermal profile displays the holding and cycling stages shown below If necessary add a step or click a temperature or time setting then change it 4 Click Next 2D Set Up Run Method Run Method Help 63 Instructions Review the reaction volume and the thermal profile for the default run method If needed edit the default run method or select a run method from the library 1 Graphical View 2 Reaction Volume Per Well E uL Expert Mode Essen A OSE Ee Holding S
142. presence absence experiments abbreviation for internal positive control IPC In the 7500 7500 Fast system software the task for the IPC target in wells that contain the IPC and do not contain IPC blocking agent See also internal positive control IPC Reagent added to PCR reactions to block amplification of the internal positive control IPC See negative control IPC wells TaqMan Gene Expression Assays or TaqMan SNP Genotyping Assays that are manufactured at the time of order Only assays that pass manufacturing quality control specifications are shipped An analysis setting in which you enter the baseline start and end values for the amplification plot You can apply the manual baseline setting to specific wells in the reaction plate manual C An analysis setting in which you enter the threshold value and select whether to use automatic baseline or manual baseline values The software uses the baseline and the threshold values to calculate the threshold cycle Cy Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 113 Experiments Glossary melt curve melt curve stage melting temperature Tm multicomponent plot negative control NC negative control blocked IPC wells negative control IPC wells no amplification control NAC no template control NTC nonfluorescent quencher minor groove binder NFQ MGB normalized quantity normalized reporte
143. pter 2 Design the Standard Curve Experiment oe Review the Reaction Setup Print Reaction Setup Instructions Print detailed reaction setup instructions then save the instructions for Chapter 3 Prepare the Reactions 1 Click Print Reaction Setup Reaction Setup Help e selectthe assay type if using TaqMan reagents then review the calculated volumes for preparing the standard dilution series edit the reaction volume excess reaction volume component concentrations and or stock concentrations Click Print Reaction pare the PCR reactions lution Calculations 6 6 ingiuL v Print Reaction Setup 1 Sample Volume uL Diluent Volume uL Total Volume of Diluted Sample uL 15 15 2 Inthe dialog box select e Detailed Reaction Setup Instructions e Include Plate Layout e Use sample color 3 Click Print to print the reaction setup instructions Print Reaction Setup Instructions Q Selectthe type of instructions to print Summary Reaction Setup Instructions Detailed Reaction Setup Instructions 9 include Plate Layout Plate Layout Print Options Use sample color Use task color 3 Preview Print Cancel 4 Click Next Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 35 Experiments man Chapter 2 Design the Standard Curve Experiment Review the Reaction Setup Design Guideli
144. quencher minor groove binder NFQ MGB can be used as the quencher With SYBR Green reagents no quencher 1s used 115 Glossary QuickStart R2 value ramp ramp speed raw data plot reaction mix reagents real time PCR reference sample refSNP ID regression coefficients 116 A feature in 7500 7500 Fast systems that allows you to run an experiment without entering plate setup information Regression coefficient calculated from the regression line in the standard curve The R value indicates the closeness of fit between the standard curve regression line and the individual Cy data points from the standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points The rate at which the temperature changes during the instrument run For the melt curve step the ramp can also be defined as a temperature increment In the graphical view of the thermal profile the ramp is indicated by a diagonal line Speed at which the temperature ramp occurs during the instrument run Available ramp speeds include fast and standard e For optimal results using the fast ramp speed Applied Biosystems recommends using TaqMan Fast reagents in your PCR reactions e For optimal results using the standard ramp speed Applied Biosystems recommends using standard reagents in your PCR reactions IMPORTANT TaqMan Fast reagents are not supported for genotyping or presence absence experimen
145. r Rn omit well outlier 114 A plot of data collected during the melt curve stage Peaks in the melt curve can indicate the melting temperature Tm of the target or can identify nonspecific PCR amplification In the 7500 7500 Fast system software you can view the melt curve as normalized reporter Rn vs temperature or as derivative reporter Rn vs temperature Also called dissociation curve In the thermal profile a stage with a temperature increment to generate a melt curve In melt curve experiments the temperature at which 50 of the DNA is double stranded and 50 of the DNA is dissociated into single stranded DNA The Tm is displayed in the melt curve A plot of the complete spectral contribution of each dye for the selected well s over the duration of the PCR run In the 7500 7500 Fast system software the task for targets or SNP assays in wells that contain water or buffer instead of sample No amplification of the target should occur in negative control wells Previously called no template control NTC In presence absence experiments wells that contain IPC blocking agent instead of sample in the PCR reaction No amplification should occur in negative control blocked IPC wells because the reaction contains no sample and amplification of the IPC 1s blocked Previously called no amplification control NAC In presence absence experiments wells that contain IPC template and buffer or water instead of sample O
146. r Analysis and Other Purposes EMC This instrument meets European requirements for emission and immunity EMC Directive 2004 108 EC This instrument has been tested to and complies with standard EN 61326 Group 1 Class B Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements This instrument has been tested to and complies with standard AS NZS 2064 Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 1 Get Started This chapter covers EH About the 7500 7500 Fast System 0 0 0 00 eens 2 E Supported Consumables go ee 4 bx ous a dais ood ee ewe bee pd a ad ad A EH About Standard Curve Experiments 0 0 0 0 0 0 eee eens 7 E How to Use This Guide 2 0 eee eens 11 m About the Example Standard Curve Experiment 0 0 00 0 00 eee 12 E Example Experiment Wars hh oes epa dp Sd OOS Se SES 14 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help 7500 Software Help Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 1 Experim
147. r Mix and RT PCR Reagents Protocol 4310251 TaqMan Drug Metabolism Genotyping Assays Protocol 4362038 TaqMan Exogenous Internal Positive Control Reagents Protocol 4308335 TaqMan Fast Universal PCR Master Mix 2X Protocol 4351891 TaqMan Gene Expression Assays Protocol 4333458 TaqMan Gene Expression Master Mix Protocol 4371135 TaqMan Genotyping Master Mix Protocol 4371131 TaqMan SNP Genotyping Assays Protocol 4332856 TaqMan Universal PCR Master Mix Protocol 4304449 User Bulletin 2 Relative Quantitation of Gene Expression 4303859 Using TaqMan Endogenous Control Assays to Select an Endogenous Control 127AP08 for Experimental Studies Application Note Note For more documentation see How to Obtain Support on page xi X Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface How to Obtain Support Obtaining The 7500 Software Help describes how to use each feature of the user interface Open Information from the Help from within the software by doing one of the following the Software Help Press FL e Click in the toolbar e Select Help gt 7500 Software Help To find topics of interest in the Help e Review the table of contents Search for a specific topic Search an alphabetized index Send Us Your Applied Biosystems welcomes your comments and suggestions for improving its user Comments documents You can e mail your comments to techpubs applie
148. r example if the diluted sample concentration is 50 0 ng uL 10X the final sample concentration in the reaction is 5 ng uL 1X See serial factor See melt curve See amplification efficiency EFF A target or gene that should be expressed at similar levels in all samples you are testing Endogenous controls are used in relative standard curve and comparative Cy AAC experiments to normalize fluorescence for the target you are quantifying Housekeeping genes can be used as endogenous controls See also housekeeping gene See post PCR read Refers to the entire process of performing a run using the 7500 7500 Fast system including setup run and analysis The types of experiments you can perform using the 7500 7500 Fast systems e Quantitation standard curve e Quantitation relative standard curve e Quantitation comparative C AAC e Melt curve e Genotyping e Presence absence Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments experiment name experiment type forward primer holding stage housekeeping gene internal positive control IPC inventoried assays IPC IPC blocking agent IPC made to order assays manual baseline Glossary Entered during experiment setup the name that is used to identify the experiment Experiment names cannot exceed 100 characters and cannot include any of the following characters forward slash ba
149. rary region of interest ROI calibration stage standard 118 In the 7500 7500 Fast system software a collection of samples The Sample Library contains the sample name and the sample color In the 7500 7500 Fast system software a reaction component displayed on the Reaction Mix Calculations tab of the Reaction Setup screen The software assumes the sample or standard 1s added to the reaction mix at a 10X concentration For example 1f the reaction volume is 20 uL the calculated volume of sample or standard for 1 reaction is 2 WL In genotyping experiments the combination of which sample to test and which SNP assay to perform in one PCR reaction Each PCR reaction can contain only one sample and one SNP assay In quantitation experiments the combination of which sample to test and which target to detect and quantify in one PCR reaction In the Design Wizard you can detect and quantify only one target in one PCR reaction Use Advanced Setup to detect and quantify more than one target in one PCR reaction In the 7500 7500 Fast system software a numerical value that defines the sequence of quantities in the standard curve The serial factor and the starting quantity are used to calculate the standard quantity for each point in the standard curve For example if the standard curve is defined with a serial factor of 1 10 or 10X the difference between any 2 adjacent points in the curve is 10 fold See standard dilution series
150. rated by the experiment data If a flag does not appear in the experiment its frequency is 0 If the frequency is gt 0 the flag appears somewhere in the experiment the well position is indicated in the Wells column Flag Description AMPNC Amplification in negative control BADROX Bad passive reference signal BLFAIL Baseline algorithm failed CTFAIL C algorithm failed EXPFAIL Exponential algorithm failed HIGHSD High standard deviation in replicate group MTP Multiple Tm peaks Note This flag is displayed only if the experiment includes a melt curve NOAMP No amplification NOISE Noise higher than others in plate NOSIGNAL No signal in well OFFSCALE Fluorescence is offscale OUTLIERRG Outlier in replicate group SPIKE Noise spikes THOLDFAIL Thresholding algorithm failed Analysis When you analyze your own standard curve experiment Guidelines e Click each flag in the Flag Details table with a frequency gt 0 to display details about the flag If needed click the troubleshooting link to view information on correcting the flag e You can change the flag settings Adjust the sensitivity so that more wells or fewer wells are flagged Change the flags that are applied by the 7500 software For More For more information on the QC Summary screen or on flag settings open the 7500 Information Software Help by clicking or pressing F1 Notes Applied Biosystems 7500 75
151. rd HE Unknown BB Unknown Flagged a 8 Check the Cy values a Select the View Well Table tab b In the Group By drop down list select Replicate c Observe the values in the Cy column In the example experiment the C values are within the expected range gt 8 and lt 35 8a rer Pete tae View ot Table 8b Select Wells With Selectitem v Show in Table W r o Expand Eoi Sollapse All ia 8c alwei Omt l pig Sample TargetN Task Dyes or Cream l crsD ouanty Quant Quant RNase P TAMRA NTC a d d d RNase P TAMRA STANDARD d d d RNase P TAMRA STANDARD d d a RNase P TAMRA STANDARD d d d RNase P TAMRA STANDARD d d Notes 12 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment m View the Standard Curve Analysis When you analyze your own standard curve experiment look for Guidelines e Slope amplification efficiency values The amplification efficiency is calculated using the slope of the regression line in the standard curve A slope close to 3 3 indicates optimal 100 PCR amplification efficiency Factors that affect amplification efficiency are The range of standard quantities For more accurate and precise efficiency measurements use a broad range 10 to 10 fold of standard quantities The number of standard replicates For more accurate effic
152. ring the PCR reactions standard dilution series and sample dilutions If needed you can edit the reaction volume excess reaction volume component concentrations standard concentration and or diluted sample concentration IMPORTANT Perform the steps above for each target assay in the reaction plate About the In the standard curve example experiment Example Experiment Applied Biosystems RNase P assay is used e The reaction volume per well is 25 uL The excess reaction volume is 10 The reaction components are TaqMan Universal PCR Master Mix 2X or TaqMan 2X Universal PCR Master Mix No AmpErase UNG RNase P Assay Mix 20X Sample or standard Water The standard concentration stock is 20 000 copies uL The diluted sample concentration is 6 6 ng UL The sample concentration stock is 100 ng uL Complete the Complete the Reaction Mix Calculations Tab see page 33 Reaction Setup Screen 1 2 3 4 5 6 Notes Select the Reaction Mix Calculations tab default In the Select Target pane select RNase P TAMRA default In the Assay Type drop down list select Inventoried Made to Order default Make sure the Reaction Volume Per Well field displays 25 uL Make sure the Excess Reaction Volume field displays 10 default In the Reactions for RNase P TAMRA pane a Make sure the Master Mix Concentration field displays 2 0X default b Make sure the Assay
153. s Real Time PCR Systems including e An introduction to TaqMan and SYBR Green reagents e Descriptions and design guidelines for the following experiment types Quantitation experiments Genotyping experiments Presence absence experiments Intended for laboratory staff and principal investigators who perform experiments using the 7500 7500 Fast system Applied Biosystems 7500 7500 Fast Real Explains how to prepare your site to receive and install the 4387776 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Vil Preface How to Use This Guide Guide Purpose and Audience PN Time PCR Software v2 0 Help Applied Biosystems 7500 7500 Fast Real Explains how to use the 7500 software to NA e Set up run and analyze experiments using the 7500 7500 Fast system e Calibrate a 7500 7500 Fast instrument e Verify that the performance of a 7500 7500 Fast instrument with an RNase P run Intended for e Laboratory staff and principal investigators who perform experiments using the 7500 7500 Fast system e Laboratory staff responsible for the installation and maintenance of the 7500 7500 Fast system Assumptions This guide assumes that you Are familiar with the Microsoft Windows operating system Are familiar with the Internet and Internet browsers Know how to handle DNA and or RNA samples and prepare them for PCR Understa
154. s eed dd 26 DS 5 5 6k sh ho 8 9 di ds O Rb 6458 dd dt 28 M Set Up the Run Method 0 0 0 0 30 E Review ihe R action DMD oo 5460 6054 ada sed dee ewe hes epi ga 245 32 E Order Materials for the Experiment 2 5440 4 45900 4i0teneed ada da da os J E Finish the Design Wizard 4 0 6 0 e nc eeae ed edna sneer ene deen cud wena en 40 Note For more information about any of the topics discussed in this guide open the Help from within Applied Biosystems 7500 7500 Fast Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help gt 7500 Software Help Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 17 Experiments in Chapter 2 Design the Standard Curve Experiment Chapter Overview Chapter Overview Notes Example Experiment Workflow This chapter explains how to use the Design Wizard in the 7500 software to set up the standard curve example experiment The Design Wizard guides you through Applied Biosystems recommended best practices as you enter design parameters for the example experiment Note When you design your own experiments you can select alternate workflows see Using This Guide with Your Own Experiments on page 11 Start Experiment v Design the Experiment Chapter 2 Create a new experiment Define the experiment properties Define the methods and materials Set up the targets Set up the samples
155. se awe GAC ag beh eee RUN ee mae ee he a ae eS 83 Section 5 2 Troubleshoot If Needed 0 cee es 85 View the Analysis Settings 0 0 ccc eee eee 86 View the OC SUMA ss 24232ci n5e2e 25025 2 eee LS E PES dE 88 Omit Wells from the Analysis 0 0c cc eee eens 90 View the Multicomponent Plot 2 0 00 ees 92 View the Raw Data Plot 0 0 0c cc ees 94 Appendix A Alternate Experiment Workflows 0 05 97 Advanced Setup Workflow 0 0 cc ee ee eee eee 98 QuickStart WORTIOW oa sega bh Mine Gata LEE 2544450005 E a 100 Template Workflow sia qc E AGE See Need ane a aha rea aoe Ae ae GO ae a eee 102 ExportImport WOrKilOW sas ss aoe es Sata eh ee ee Pee Bae 104 iv Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments BIDIIOOra DNY 44 0022 seemed a tae SR oe eee ee Glossary Meese ate eee ee Sea ears Ge ere era se are Gees Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments vi Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface How to Use This Guide About the System The guides listed below are shipped with the Applied Biosystems 7500 7500 Fast Documentation Real Time PCR System Time PCR System Site Preparation Guide 7500 7500 Fast system Intended for p
156. so that the 7500 software alerts you by e mail when the 7500 7500 Fast instrument begins and completes the run or 1f an error occurs during the run Enabling the notifications settings 1s optional and does not affect the performance of the 7500 7500 Fast system or the duration of the run IMPORTANT The notification settings feature is available only if the computer that you are using 1s running the 7500 7500 Fast instrument and 1s connected to an Ethernet network In the example experiment The 7500 software is set up to send notifications to three users scientist supervisor and technician at mycompany com when the 7500 7500 Fast system ends the run and if it encounters any errors during operation e The example SMTP server www mycompany com is set up for secure sockets layer SSL encryption and requires authentication for use 1 In the 7500 software click wn Run in the navigation pane Click E Notification Settings Select Yes for Enable Notifications gt P N Select the events that will generate notifications a Select Instrument Error b Select Run Completed 5 In the Enter e mail addresses for notifications field enter scientist mycompany com supervisor mycompany com technician mycompany com 6 In the Outgoing Mail Server SMTP field enter smtp mycompany com f Set the authentication settings a Select Yes for Server requires authentication b In the User Name field enter Example User c
157. spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Preface Chemical Waste Safety Chemical Waste Safety Chemical Waste Near HAZARDOUS WASTE Refer to Material Safety Data Sheets and Hazard local regulations for handling and disposal Nem CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death AN WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical Waste To minimize the hazards of chemical waste Safety Guidelines Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A se
158. standard curve is set up for the target RNase P The standard used for the standard dilution series contains known quantities of the RNase P gene Because a single gene is being studied only one standard curve is required Five points are used in the standard curve Three replicates are used for each point Replicates are identical reactions containing identical reaction components and volumes The starting quantity is 10 000 copies and the serial factor is 1 2 Click the How many points do you need for each standard curve field then enter 5 Click the How many replicates do you need for each point field then enter 3 Define the range of standard quantities for the RNase P experiment a Click the Enter Starting Quantity field then enter 10000 b In the Select Serial Factor drop down list select 1 2 Review the Standard Curve Preview pane The standard curve has the following points 10000 5000 2500 1250 and 625 Click Next 2B Set Up Standards Standards Help amp Instructions Enter the number of points and replicates for all standard curves in the reaction plate For each standard curve enter the starting quantity and select the serial factor Set Up Standards Required k View Plate Layout How many points do you need for each standard curve 5 Arrange Plate by Rows v Place Negative Controls in Upper Left Ho
159. t 18 19 20 21 22 23 24 Well Omit Low Cr less than 24 Flag Medium Cr from 24 to 30 A4 A5 AG AT A8 A9 A10 A11 A12 B1 B2 B3 B4 B5 B6 B7 B8 B9 OOOOOOOOOOOOOOOOOO High Cr greater than 30 B10 B11 B12 Undetermined A1 A2 A3 lt JOO OOO gt Sample pop1 pop1 pop1 pop pop pop Target N RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT Task UNKNOWN UNKNOWN UNKNOWN UNKNOWN UNKNOWN UNKNOWN STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD NTC NTC NTC Dyes FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA GT 28 280773 28 241549 28 259768 27 241302 27 18564 27 174326 26 389278 26 213354 26 242676 27 36778 27 258842 27 206913 28 325405 28 28138 28 69632 29 64913 29 546368 29 421392 30 557467 30 551697 30 982454 Undetermi Undetermi Undetermi CT
160. tage Holding Stage Cycling Stage NumberofCycles 40 C Enable AutoDelta Starting Cycle E 4 3 Legend Data Collection On Data Collection Off A AutoDeltaOn A AutoDelta Off Design When you design your own standard curve experiment Guidelines e Enter a reaction volume well Applied Biosystems recommends a reaction volume of 25 uL for standard curve experiments The 7500 system supports reaction volumes from 20 to 100 uL The 7500 Fast system supports reaction volumes from 10 to 30 UL Review the thermal profile Make sure the thermal profile is appropriate for your reagents If you are performing 1 step RT PCR include a reverse transcription step If your experiment requires a different thermal profile edit the thermal profile or replace the run method with one from the Run Method library The Run Method library is included in the 7500 software For More For more information on Information The Run Method library or on completing the Run Method screen Open the 7500 Software Help by clicking or pressing F1 e Using Advanced Setup See Advanced Setup Workflow on page 98 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 31 Experiments O Chapter 2 Design the Standard Curve Experiment Review the Reaction Setup Review the Reaction Setup In the Reaction Setup screen select the assay type if using TaqMan reagents then review the calculated volumes for prepa
161. tem Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment ss Omit Wells from the Analysis 6 Click Analyze to reanalyze the experiment data with the outlying well s removed from the analysis f Review the data analyzed without the outlier Standard Curve a In the navigation pane select Analysis gt b Display all 96 wells in the Standard Curve screen by clicking the upper left corner of the plate layout in the View Plate Layout tab c In the Target drop down list select All d In the Plot Color drop down list select Default 8 View the values displayed below the standard curve In the example experiment the values for the target RNase P are within the acceptable ranges Slope is 3 614 R value is 0 993 Amplification efficiency Eff is 89 118 Analysis When you analyze your own standard curve experiment carefully view the replicate Guidelines groups for outliers If needed remove outliers manually using the Well Table For More For more information on omitting wells from the analysis open the 7500 Software Help Information by clicking or pressing F1 Within the Help search for the omit well topics 1 Select the Search tab 2 Enter omit well 3 Click List Topics 4 Double click the topics you want to review Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 91 Experiments ma
162. the part number link You are connected to the product information screen in the Applied Biosystems Store To open the Applied Biosystems Store you must have an Internet connection 3 Optional Click Print Materials List to print the materials list Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 37 Experiments alee Chapter 2 Design the Standard Curve Experiment Order Materials for the Experiment 4 Optional Create a shopping list a Select the check box next to each of the following items e MicroAmp Optical 96 Well Reaction Plate e MicroAmp Optical Adhesive Film e MicroAmp Splash Free Support Base e TaqMan Universal PCR Master Mix 2X or TaqMan Universal PCR Master Mix No Amperase UNG b Click Add Selected Items to Shopping List 5 Optional Create a shopping basket in the Applied Biosystems Store Product availability and pricing may vary according to your region or country Online ordering through the Applied Biosystems Store is not available in all countries Contact your local Applied Biosystems representative for help a Check that the Experiment Shopping List contains the desired materials and that the quantities are correct then click Order Materials in List b In the Order Materials Log In dialog box enter your user name and password for the Applied Biosystems Store then click Log In and Submit Note If you do not have an account
163. tify in the reaction plate field then enter 1 Note The number of rows in the targets table is updated with the number you entered Select the Set Up Standards check box to set up standards for the target experiment Note The Set Up Standards check box is selected by default Set up the Target 1 experiment a Click the Enter Target Name cell then enter RNase P TAMRA b In the Reporter drop down list select FAM default c In the Quencher drop down list select TAMRA d In the Color field leave the default 24 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment a Set Up the Targets 4 Click Next Note Leave blank the Optional Enter Gene Name field You can search for the gene assay ID when you order your materials see Order Materials for the Experiment on page 37 2A Set Up Targets Targets Help instructions Enter the number of targets to quantity in the reaction plate then set up the assay for each target Set Up Targets Required 1 How many targets do you want to quantify in the reaction plate 1 fee et gure rarer IS ET ea Pan a ES Set Up Standar Enter Target Name Reporter Quencher Color AssayID Optional Enter Gene Name and Click Fi 3a 3b 3c 3d Design When you design your own standard curve experiment Guidelines e
164. tifying information for the experiment select the instrument type then select the type of experiment to design About the In the standard curve example experiment Example r h The experiment is identified as an example Experiment e The instrument that is selected to run the experiment is the 7500 instrument e A MicroAmp Optical 96 Well Reaction Plate is used e The experiment type is quantitation Complete the 1 Click the Experiment Name field then enter Standard Curve Example Experiment Properties Note The experiment header is updated with the experiment name you entered Screen 2 Leave the Barcode field empty 3 Click the User Name field then enter Example User 4 Click the Comments field then enter Standard Curve Getting Started Guide Example 5 Select 7500 96 Wells 6 Select Quantitation for the experiment type T Click Next 1A Define Experiment Properties Experiment Properties Help 3 Instructions Enter an experiment name selectthe instrument type then select the type of experiment to design How do you want to identify this experiment Required ERPE TENNIS l Standard Curve Example ee ee eee LIGIA NS pI Use Wane wpuunaly Example User KR OND a Comments Ontinnal Standard Curve Getting Started Guide Example lei Which instrument are you using to run the experiment Set up run and analyze an experime
165. ts A plot of raw fluorescence not normalized for each optical filter A solution that contains all components to run the PCR reaction except for the template sample standard or control The PCR reaction components you are using to amplify the target and to detect amplification Types of reagents used on the 7500 7500 Fast systems e TaqMan reagents e SYBR Green reagents e Other reagents Process of collecting fluorescence data during PCR Data from the real time PCR are used to calculate results for quantitation experiments or to troubleshoot results for genotyping or presence absence experiments In relative standard curve and comparative Cy AAC experiments the sample used as the basis for relative quantitation results Also called the calibrator Identifies the reference SNP refSNP cluster ID Generated by the Single Nucleotide Polymorphism Database of Nucleotide Sequence Variation dbSNP at the National Center for Biotechnology Information NCBI The refSNP ID can be used to search the Applied Biosystems Store for an Applied Biosystems SNP Genotyping Assay Also called an rs number Values calculated from the regression line in standard curves including the R value slope and y intercept You can use the regression coefficients to evaluate the quality of results from the standards See also standard curve Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve
166. ts to generate standard curves See also standard curve and standard dilution series Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments standard curve standard curve method standard dilution series standard quantity starting quantity Glossary In standard curve and relative standard curve experiments e The best fit line in a plot of the C values from the standard reactions plotted against standard quantities See also regression line A set of standards containing a range of known quantities Results from the standard curve reactions are used to generate the standard curve The standard curve is defined by the number of points in the dilution series the number of standard replicates the starting quantity and the serial factor See also standard dilution series Method for determining absolute target quantity in samples With the standard curve method the 7500 7500 Fast system software measures amplification of the target in samples and in a standard dilution series Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates the absolute quantity of target in the samples See also standard and standard curve In standard curve and relative standard curve experiments a set of standards containing a range of known quantities The standard dilution series is prepared by serially diluting stan
167. ttings Default CT settings are used to calculate the Cr for targets without custom settings To edit the default settings click Edit Default Settings Threshold AUTO Baseline Start Cycle AUTO Baseline End Cycle AUTO Edit Default Settings Select a Target Cr Settings for RNase P TAMRA Target Threshold Baseline Start Baseline End CT Settings to Use Use Default Settings RNase P TAMRA AUTO AUTO AUTO A Revert to Default Analysis Settings Apply Analysis Settings Cancel Notes 86 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment m tico View the Analysis Settings Analysis Unless you have already determined the optimal settings for your experiment use the Guidelines default analysis settings in the 7500 software If the default settings are not suitable for your experiment you can change the settings Use the e Cy Settings tab to manually set the threshold and baseline When manually setting the threshold and baseline Applied Biosystems recommends the following Setting Recommendation Threshold Enter a value for the threshold so that the threshold is e Above the background e Below the plateau and linear regions of the amplification curve e Within the exponential phase of the amplification curve Baseline Select the Start Cycle and End Cycle values so that the baseline ends before significant fluorescence is detected
168. tware updates the Run Status field throughout the run The figure below shows the Run Method screen as it appears in the example experiment To Action A Change the number of cycles In the Extend of Cycles field enter the number of cycles to apply to the Cycling Stage B Apply your changes Click Send to Instrument Run Method Edit Run Method ee stage is a cycling stage that collects data Jur Enable AutoDelta Starting Cycle Extend of Cycles 1 Send to Instrument E Legend Data Collection On Data Collection Off A AutoDelta On A AutoDelta Off If an alert appears click the error for more information then troubleshoot the problem as explained in the 7500 Software Help click or press F1 Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 63 Experiments wr Chapter 4 Run the Experiment Unload the Instrument Unload the Instrument When your 7500 7500 Fast system displays the Run Complete message unload the reaction plate from the instrument Unload the PAIN WARNING PHYSICAL INJURY HAZARD During operation the sample Reaction Plate block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature 1 Push the tray door to open 1t 2 Remove the reaction plate 3 Push the tray door to close it
169. ty xx electromagnetic compatibility standards See EMC standards EMC standards xxii ergonomics safety xxi example experiment analyze 66 description 12 design 18 name 20 prepare 44 run 56 workflow 14 Experiment Properties screen 20 EXPFAIL flag 89 Export Import 11 104 F flag 83 flags analysis settings 87 in standard curve experiments 89 G guidelines analysis 69 73 78 83 87 89 91 94 95 chemical safety xviii chemical waste disposal xix chemical waste safety xix design 21 23 25 27 29 31 36 39 42 preparation 46 48 50 53 run 60 H hazard icons See safety symbols on instruments hazard symbols See safety symbols on instruments hazards See safety Help system accessing xi HIGHSD flag 89 IMPORTANT description xii installation category xx instrument operation safety xvi Inventoried assays 36 L library 31 load reaction plate 57 M Made to Order assays 36 Materials List screen 37 materials required 45 47 49 52 Methods amp Materials screen 22 monitor run Amplification Plot screen 62 Run Method screen 63 moving and lifting safety xvi moving parts safety xx MSDSs description xvii obtaining xvii MSDSs obtaining xi MTP flag 89 Multicomponent Plot screen 92 Multiple Plots screen 70 multiplex PCR 8 24 N navigation tips display multiple plots 70 select wells 69 negative control component of experiment 7 NOAMP flag 89 NOISE flag 89 NOSIGNAL flag 89
170. u can either Accept the plate layout automatically generated by the software or Use Advanced Setup to change the plate layout in the software Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 53 Experiments 2 Chapter 3 Prepare the Reactions Prepare the Reaction Plate e If you use optical adhesive film seal each reaction plate as follows a b C Place the reaction plate onto the center of the 96 well base Load the reaction plate as desired Remove a single optical adhesive film film from the box Fold back one of the end tabs Hold the film with its backing side up In one continuous movement peel back the white protective backing from the center sealing surface Do not touch the center sealing surface IMPORTANT Improper peeling of the optical adhesive film may result in haziness but does not affect results Haziness disappears when the film comes into contact with the heated cover in the instrument While holding the film by the end tabs lower the film onto the reaction plate adhesive side facing the reaction plate ZS Be sure the film completely covers all wells of the reaction SS S plate A While applying firm pressure move the applicator slowly across the film horizontally and vertically to ensure good contact between the film and the entire surface of the reaction plate N While using the applicator to hold th
171. ve Applied Biosystems recommends three replicates for each point e Because the range of standard quantities affects the amplification efficiency calculations carefully consider the appropriate range of standard quantities for your assay For more accurate measurements of amplification efficiency use a broad range of standard quantities such as between 10 and 10 If you specify a broad range of quantities for the standards you need to use a PCR product or a highly concentrated template such as a cDNA clone If you have a limited amount of cDNA template and or if the target is a low copy number transcript or known to occur within a given range a narrow range of standard quantities may be necessary e The serial factor is used to calculate the quantities in all points of the standard curve If your starting quantity is the highest quantity select a dilution factor such as 1 2 1 3 and so on If your starting quantity is the lowest quantity select a concentration factor such as 2X 3X and so on For More For more information on Information e Completing the Standards screen Open the 7500 Software Help by clicking or pressing F1 e Amplification efficiency Refer to the Amplification Efficiency of TaqMan Gene Expression Assays Application Note Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 2 Experiments in Chapter 2 Design the Standard Curve
172. w many replicates do you need for each point 4 For each standard curve define the range of standard quantities by entering the starting quantity and serial factor Define the standard curve so that the range of standard quantities spans the expected R p Sam Sam Sa m Sam Sam Sam R range of quantities for the unknowns A N ii IN IN p m mr fy p m w R gF Ss Target Name Enter Starting Quantity Select Serial Factor SUSU SSeS ee esse eeu es eeeeueeeseeeeeesseseseessuseeusseeseuusseeusuueseeseuees R R nasais AA E TE J AS is bal Sha al S aS Ms aS BLS Ma s BLS NS v era cr era AQerer rer 2 er 4 a ane enc ene ene Standard Curve Preview C D E F G H M 6 Unknown 9 15 Standard 0 3 Negative Control 72 Empty 26 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 2 Design the Standard Curve Experiment O Set Up the Standards Design When you design your own standard curve experiment Guidelines e Setup a standard curve for each target in the reaction plate The targets are previously defined on the Targets screen Set Up the Targets on page 24 e Enter the number of points for each standard curve in the reaction plate Applied Biosystems recommends at least five dilution points for each standard curve e Enter the number of replicates identical reactions for each point in the standard cur
173. wo rows 4 To display two plots in columns click Show plots in two columns 5 To display a specific plot select the plot in the drop down list above each plot display 5 Multiple Plots View o Amplification Plot ARn vs Cycle v PL att E a BE Standard Curve Amplification Plot 0 000001 1a 22 2 M A wn az Ed E v a Legend Standard H Unknown ogend A E B a c E D E Unknown Flagged Multicomponent Plot Raw Data Plot Cycle Multicomponent Plot Raw Data Plot 2 500 000 Fluorescence m nl n pP Legend g TAMRA pm FAM gE af n gt Notes 70 Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve Experiments Chapter 5 Analyze the Experiment ms View the Standard Curve View the Standard Curve About the Example Experiment View the Standard Curve Notes In the navigation pane select Analysis gt The Standard Curve screen displays the standard curve for samples designated as standards The 7500 software calculates the quantity of an unknown target from the standard curve In the standard curve example experiment you review the Standard Curve screen for the following regression coefficient values e Slope amplification efficiency e R value correlation coefficient e Cy values Note In the example experiment the slope R and amplificatio
174. xperiments Appendix A Alternate Experiment Workflows A Advanced Setup Workflow A 4 Prepare the PCR reactions Experiment Type Prepare the Relative standard curve Standard curve Template Sample dilutions Standard dilution series Reaction mix Reaction plate o OOo Too Comparative Cr a Template Genotyping b Sampie Cuong c Reaction mix Presence absence d Reaction plate 5 Run the experiment IMPORTANT While the 7500 7500 Fast instrument is performing a run do not create experiments perform maintenance or allow the computer to run antivirus software or to enter hibernation Performing such activities while the instrument is running an experiment will cause gaps in data collection Load the reaction plate into the instrument a b Start the run O Optional Monitor the run Q Unload the reaction plate from the instrument 6 Analyze the data Q Open the experiment in the 7500 software oO In the navigation pane click Analysis c If the data are not analyzed click Analyze d In the navigation pane select an analysis screen to view the data for example select QC Summary to view a quality summary of the data Notes Applied Biosystems 7500 7500 Fast Real Time PCR System Getting Started Guide for Standard Curve 99 Experiments A Appendix A Alternate Experiment Workflows ER QuickStart Workflow QuickSt
175. yze the Experiment 2b 2C Notes View the Well Table b In the Group By drop down list select Flag The software groups the flagged and unflagged wells In the example experiment well B12 is flagged View Plate Layout Show in Table View Well Table Group By Select Wells With l Select Item Select Item Expand All Collapse All _ Well Flagged Wells B12 Omit b Unflagged Wells A1 A2 A3 A4 A5 AG AT A8 A9 A10 A11 A12 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 C1 C JOOOOOOOOOOOOOOOOOOOOOOOO c In the Group By drop down list select Cy The software groups the wells by Cy value low medium high and undetermined In the example experiment View Plate Layout Show in Table W Flag Sample pop1 pop1 popi pop2 pop2 pop2 Target N RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT RNase PT Task STANDARD NTC NTC NTC UNKNOWN UNKNOWN UNKNOWN UNKNOWN UNKNOWN UNKNOWN STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD STANDARD Dyes FAM TAMRA FAM TAMRA FAM TAMRA FAM TAMRA

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