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Applied Biosystems 7300/7500 Real Time PCR System Plus/Minus

1. n n Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknow m Dc Ow Ow Ow Ow Ow Ow Ow m m m G Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknow no mo o o o o o o o mo nno o o Do Dc Dc Dc Dc o Ow o no L Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknow Ready Connected 9730 8000 6000 Fluorescence 4000 2000 697 Filters Cycle 40 40 1 Cycle Plus Minus Getting Started Guide for the 7300 7500 System o3 Appendix B Viewing the Amplification Data Component Tab This tab displays the complete spectral contribution of each dye in a selected wells over the duration of the PCR run Only the first selected well is shown at one time Double clicking the y axis displays the Graph settings dialog box Amplification Plot Tab The Amplification Plot tab allows you to view both real time and post run amplification of specific samples The Amplification Plot tab displays all samples in the selected wells Rn vs Cycle Linear This plot displays normalized reporter R dye fluorescence as a function of cycle You can use this plot to identify and examine irregular amplification For more information about R
2. cee eee Creating Detectors Viewing Amplification Data Specifying Analysis Settings 0 0 0 ee eee Analyzing the Plus Minus Amplification Data AQ Plate Viewing the Amplification Data llle References Index Plus Minus Getting Started Guide for the 7300 7500 System Preface How to Use This Guide Purpose of This Guide Assumptions Text Conventions User Attention Words Safety This manual is written for principal investigators and laboratory staff who run plus minus assays using the Applied Biosystems 7300 7500 Real Time PCR System 7300 7500 system This guide assumes that you have Familiarity with Microsoft Windows XP operating system Knowledge of general techniques for handling DNA samples and preparing them for PCR A general understanding of hard drives and data storage file transfers and copying and pasting If you want to integrate the 7300 7500 system into your existing laboratory data flow system you need networking experience This guide uses the following conventions Bold indicates user action For example Type 0 then press Enter for each of the remaining fields talic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu For example
3. lt lt gt Designing Use TaqMan Chapter 2 YW a Plus Minus probe based reagent j pa Experiment configuration and primers Design the probe the Reaction Chapter 3 een Prepare DNA Set up reaction plate Performing Chapter 4 USUS the Plus Minus Create a Plus Minus Perform the sini Pre Read Run plate document pre read run 4 Generating Create a plate Chapter 5 w 7 Amplification document for Data sample amplification Start the amplification run Performin l the Plus Minus Perform a View Export plate Post Read Run post read run Plus Minus results documents Chapter 6 Plus Minus Getting Started Guide for the 7300 7500 System Example Plus Minus experiment Plus Minus Experiment Workflow Plus Minus Getting Started Guide for the 7300 7500 System Chapter 1 Chapter 2 Chapter 3 Chapter 4 Contents Plus Minus Experiment Workflow lii Preface vil How to Use This Guide d x ccs dex acc a ere ae ee eee eee ge ee ew Rs AP Bes vil How to Obtain More Information 0 00 ccc eee ees Viii How to Obtain Services and Support llle viii Send US YOuUrCOMMENIS 204 Qeidiicdpee eue Dame dw qi dob Yo D dre de E p dl odes viii Introduction 1 Sal M PPD r 1 About th 7300 7500 Syst m cs uos de ace aia cha be EO cee eR WU d a edu pas ves 2 About Plus Minus Assays Using an IPC
4. 38 Plus Minus Getting Started Guide for the 7300 7500 System Performing the Plus Minus Post Read Run is ca E wn Urn De n Chapter 6 3 HN HN EN Workflow Perform a post read run See page 40 View Plus Minus results See page 43 Export plate documents See page 47 Performing the Plus Minus Post Read Run Notes Plus Minus Getting Started Guide for the 7300 7500 System 39 Performing the Post Read Run Chapter 6 Performing the Plus Minus Post Read Run Performing the Post Head Run Open the pre read plate document 1 Select the Instrument tab 2 Accept the defaults for e Sample volume 50 uL e 9600 Emulation selected Note The 9600 Emulation feature is not available for the 7300 instrument 3 Select File gt Save As type the name Plus Minus Post Read for the plus minus plate document then click Save Notes Setup Y Instrument Y Results X m Instrument Control Temperature PreRead Estimated Time Remaining hh mm Sample Heat Sink Cover Block Cycle Disconnect Status Stage Rep Idle Time rnm ss Step State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Add Cycle Add Hold Add Step Add Dissociation Stage HI tnt Sample Volume pL 50 Iv 9600 Emulation Amplification Plot Data Stage 3 Step 2 60 0
5. Notes 47 7 Generating o T Amplification _ Data Create a plate document d c See page 32 for sample amplification Start the eee See page 36 amplification run Plus Minus Getting Started Guide for the 7300 7500 System 31 Chapter 5 Generating Amplification Data Creating a Plate Document for Sample Amplification Creating a Plate Document for Sample Amplification Benefits of Because the plus minus assay is an end point assay you can amplify the target sequences Real Time offline using any thermal cycler However using the 7300 7500 system to amplify the Amplification target sequences provides real time PCR data After the plus minus samples are analyzed you can study the amplification plots 1f you observe questionable calls or observe no data for a well Using AQ Plate You create and use absolute quantification AQ plate documents to store real time data Documents for for plus minus assays Because the AQ plate document is used only to amplify target Amplification sequences not to quantify the PCR data you do not need a standard curve for the AQ plate Detector Tasks For AQ plate documents there are three types of tasks Task Symbol Apply to Unknown ul Detectors of wells that contain target sequences Standard E Detectors of wells that contain samples of known quantities NIC M Detectors of negative control wells that contain no template The ta
6. Read se eee Plus Minus Pre Read 5 Click Next gt Finish Cancel Notes 24 Plus Minus Getting Started Guide for the 7300 7500 System 6 Select the detectors to add to the plate document a Click to select a detector Ctrl click to select multiple detectors If no detectors are listed refer to Appendix A Creating Detectors b Click Add gt gt The detector s are added to the Detectors in Document list box IMPORTANT Ensure that the reporter dye for the target is different from the reporter dye for the IPC which is VIC Note The TaqMan Exogenous Internal Positive Control Reagents Kit PN 4308323 uses an IPC VIC labeled probe with TAMRA quencher Note To remove a detector in the Detectors in Document window select the detector then click Remove c Click Next f Select six wells on the plate document for the no amplification controls blocked IPC and no target template a Select wells Al to A6 b Select the target detector by checking the Use box next to it c Select Task gt NTC An N appears in the well d Select the IPC detector by checking the Use box next to it e Select Task gt NTC A second N appears in the well Notes Creating a Plus Minus Plate Document Creating a New Plus Minus Plate Document New Document Wizard Select Detectors Select the detectors you will be using in the document Passive Reference ROX Detector Hame
7. c Type the sample name Notes Chapter 4 Performing the Plus Minus Pre Read Run New Document Wizard Set Up Sample Plate Setup the sample plate with tasks and detectors wv IPC IPC Unknown NTC IPC IPC F G H lt Back Finish Cancel New Document Wizard Setup Sample Plate Setup the sample plate with tasks quantities and detectors NTC IPC GE 0A 0 GUB UE DUE UE 0E UU uE Uum oo oo DE 0H dC 0H CH 10H UH XH YU Cao eo ee ee es eee lle Wuru ruru ME IN I IN Im Imi HN IN Im oe ooo oo LEN IN Sn J Sn en Sn en en JOE UEM a oW HN IN IN IN S IN IN IN IN IN on IU lt Back l Finish Cancel o 4 o o it hah l Fuse Detector Reporter quencher Tasi Color Iv c VIC TAMRA NTC V Ecol FAM TAMRA NTC i T o o o oO o oO oO oO o o u Oo oO 26 Plus Minus Getting Started Guide for the 7300 7500 System d Accept the default setting ROX for the Passive Reference ROX dye e Repeat steps b through c until you name all wells f Click E to close the Well Inspector IMPORTANT If your experiment does not use all the wells on a plate do not omit the wells from use at this point You can omit unused wells after the run is completed For more information about omitting wells refer to Online Help Note You can change the sample setup information sample name detector task after a run is complete g Verify the infor
8. lem Reaction Concentration Nnm CHEMICAL HAZARD TaqMan 25 0 1X TaqMan Universal PCR Master Mix may cause Universal PCR eye and skin irritation Exposure may cause Master MIX oos discomfort if swallowed or inhaled Read the 10X Exo IPC Mix 5 0 50 to 900 nM MSDS and follow the handling instructions IPC kit Wear appropriate protective eyewear clothing 50X Exo IPC 10 50 to 900 nM and gloves DNA IPC kit Target primers 14 0 50 to 250 nM probe and deionized water Total 45 0 2 Into each well pipette 45 uL of the reaction mix If preparing Then add 3 Pipette 5 uL of sample NAC NTC or unknowns into each well of a 96 well plate NAC 5 uL of 10X Exo IPC Block IPC kit Note The final reaction volume in each well NTC 5 uL of 1X TE or H O should be 50 uL a No Amplification Control well contains no target template and blocked IPC b No Template Control well contains no target template only IPC 4 Keep the reactions on ice until the plate is loaded into the 7300 7500 instrument Example Experiment For the example experiment 45 uL of reaction mix is pipetted into each well of a 96 well plate and 5 uL of the following is added as specified in the table below Wells To prepare Add to each well A1 to A6 NAC 5 uL of 10X Exo IPC Block IPC kit A7 to A12 NTC 5 uL of 1X TE or H O B1 to H12 Ua 5 uL of sample DNA a Unknown well contains both target template an
9. llle 2 About Plus Minus Assays 3 03 Sce ie alae e a e e a mde iU dC eee urs 4 Example Plus Minus Experiment eere rn 6 Designing a Plus Minus Experiment 13 liec qr teresa od encircle coe sate sake Gg oak eed dee a a Oak ert RA 13 Using TaqMan Probe Based Reagent Configuration sees 14 Designing the Probe and Primers llle 15 Setting Up the Reaction Plate 17 WOEKIOW Mer m cL ITI 17 misera pM rcv pcr 18 Setting Up the Reaction Plate llli 19 Performing the Plus Minus Pre Read Run 21 VOTO UP reni tot cts eid ah gtr seein shee onu sa ee eae utar d ett eee unes A e 21 dne Presnedu RUN Seca aa awii madre star Aa ey ge e E 6 dete aues ua Bea EAT pad Rt 22 Before You Begill ecn Sad mos Die ob aov duod dus Doduc e E No Rede 22 Creating a Plus Minus Plate Document cece eee ees 22 Performing the Pre Read Run 2 00 teens 28 Plus Minus Getting Started Guide for the 7300 7500 System V Chapter 5 Chapter 6 Appendix A Appendix B vi Generating Amplification Data WOCKIOW i aot i a iaa aa a a a ag Daler a a Bel Geshe ly a Creating a Plate Document for Sample Amplification Performing the Amplification Run 0 0 aaaea Performing the Plus Minus Post Read Run Tego PT Performing the Post Read Run 0 000 cee eee ees Viewing Plus Minus Results eee eee eee Exporting Plus Minus Plate Data
10. DOES E ee Unknown Data Delta Rn vs Cycle w RE mo Detector All Ad 0 60 HHHH MA 4 3 A 5 AM Detector Color Line Color Detector Color E coli 0 50 m Analysis Settings Auto Ct 0 40 C Manual Ct Threshold mixed IP C Well F1 0 30 fe Delta Rn C Hu 0 20 Start cycle amp uto End cycle amp uto 0 10 D 1234567 8 9101112131415161718192021 222324 25 26 27 2829 3031 32 333435 35 37 38 39 40 Cycle Number Example Experiment The image below shows the amplification plot for unknown sample well B9 which displays a questionable result The unknown sample is labeled with a question mark because the sample target threshold is below the IPC threshold but above the NAC signal Setup Y instrument Y Results Plate Y Spectra Y Component Y Amplification Plot Delta Rn vs Cycle EN l mEN Data Delta Rn vs Cycle Unknown Detector fan I Cf 0 05 H Line Color Detector Color I 0 04 _ 1L LL I LZ IL Lg lg p Analysis SEUNS IPC C Auto Ct Manual Ct 0 03 Threshold 0 092972 C Auto Baseline Well B9 a 0 02 H Manual Baseline Start cycle 3 End cycle 15 g ud ai E coli o Ea 0 01 12345 6 7 8 91011121314151617 1819 2021 22 23 24 25 26 27 28 29 3031 32 3334 35 36 37 38 39 40 Cycle Number Notes 46 Plus Minus Getting Started Guide for the 7300 7500
11. Description Reporter Quenche Detectors in Document Internal Positiv IC none none Remove none none none none none none none fnane New Detector lt Back Next gt Finish Cancel New Document Wizard Setup Sample Plate Setup the sample plate wih lacks quantities and detectors uso Detector Ra F Pc v Plus Minus Getting Started Guide for the 7300 7500 System 25 Creating a Plus Minus Plate Document 8 Select six more wells on the plate document for the no template controls IPC but no target template a Select wells A7 to A12 b Select the target detector by checking the Use box next to it c Select Task gt NTC An N appears in the well d Select the IPC detector by checking the Use box next to it e Select Task gt IPC An I appears in the well next to the N 9 Select all remaining wells for the unknown samples IPC and target template a Select wells B1 to H12 by click dragging across all empty wells b Select the target detector by checking the Use box next to it c Select Task gt Unknown A U appears in the boxes d Select the IPC detector by checking the Use box next to it e Select Task gt IPC An I appears next to the U 10 Click Finish 11 Enter sample names for each well a Click or select View gt Well Inspector from the menu b Click drag to select replicate wells
12. ERE ee ee es ee No template n No template n No template ni No template n Mo template n Mo template n No template No template No template Unknown Dc Unknown Dco Unknown mi Unknown mi Unknown m Unknown m Unknown nno peces i n ae eaa iei e ee m m m E Unknown Do Unknown mo Unknown Ow H Unknown Ow Unknown Ow Unknown Oo Unknown o Unknown nc Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown Dc Oo Unknown Oo Unknown n Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown nno Unknown no Unknown no Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown Oo Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown nno Unknown nno Unknown Ow Unknown Ow Unknown Ow Unknown Ow Unknown Ow u Unknown Oe u Unknown Ow Unknown Ow u Unknown Ow Unknown Ow Unknown Ow a Unknown Ow u Unknown nno Unknown mo Unknown mo Unknown ue Unknown mo Unk
13. No template n No template Mo template Mo template Mo template No template No template Uu INI Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown o Ute o Ute yc Do o Do Do Ute Do 1 1 1 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Do ute Dco Ute D o Ute u fes v s Ue o Ute L Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Ow o Dco c c Ow Ow Do Ow Ute o u Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Ow c UTS Dco Dco Ow o o o c Ow u u a u oD Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown ute ute ue u E ute Ow ute ute Dco Dco o Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown o c Do c Dc Ow Dc c Dco c Ow u m u Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown c o Dc Ow Oe Ow o o c o o u 1 m u Notes 42 Plus Minus Getting Started Guide for the 7300 7500 System Viewing Plus Minus Results Viewing Plus Minus Results Results After completing the plus minus pos
14. Plus Minus This document uses the term plus minus assay to refer to the entire process of Experiment analyzing samples of extracted DNA from data collected at the end of the PCR process Workflow After you design the experiment and isolate DNA a plus minus assay involves performing A pre read run on a plus minus plate document to determine the baseline fluorescence associated with primers and probes before amplification An amplification run using an AQ plate document to generate real time PCR data which can be used to analyze and troubleshoot the PCR data for the plus minus assay if needed A post read run using the original plus minus plate document which automatically subtracts the baseline fluorescence determined during the pre read run to calculate the result The following figure illustrates the complete process Reaction Plate 7300 7500 System Pre Read Run Amplification Run Post Read Run Notes 4 Plus Minus Getting Started Guide for the 7300 7500 System Required User Notes Supplied Materials About Plus Minus Assays Required User Supplied Materials Item Source DNA isolation and purification chemistry systems e ABI PRISM 6100 Nucleic Acid PrepStation e BloodPrep Chemistry genomic DNA from fresh or frozen blood or cells e NucPrep Chemistry DNA from animal
15. System Exporting Plus Minus Plate Data Note For more information on analyzing the amplification data and your plus minus results see Appendix B Viewing Amplification Data Exporting Plus Minus Plate Data You can export numeric data from plus minus plates into text files which can then be imported into spreadsheet applications such as Microsoft Excel 1 Select File gt Export then select the data type to export Sample Setup txt ELS View Tools Instrument Analysis Window Help Calibration Data csv Qo Mew CrHN e e aa P Open Ctri o e Spectra csv Close Save Chrl 5 Save Os fe ee ea te m Mo template m Mo template m Component csv Rn csv Refer to the Online Help for information about the export file types Revert To Saved Import Sample Setup D INE Export Sample Setup View Exported Results Calibration Data Page Setup Spectra Frink Preview Component Print Ctrl F Rn 1 Plus Minus Post Read Results Unknown Unknown mi mi Eli Exil 2 Type a file name for the export file Select Sample Setup Export File Note The name of the dialog box depends on the type of data you selected to export hy Recent Documents 3 Click Save lt My Network File name SampleSpectrak partFile csv Save Places Save as type Sample Setup Export File Cancel Notes Plus
16. There are three possible combinations the NN box seen below is the NAC no target template plus blocked IPC the NI box seen below is the NTC no target template plus IPC Notes Plus Minus Getting Started Guide for the 7300 7500 System 27 Performing the Pre Read Run Performing the Pre Head Run 1 Select the Instrument tab 2 Accept the default values for Sample volume 50 uL e 9600 Emulation selected Note The 9600 Emulation feature is not available for the 7300 instrument 3 Select File gt Save As type Plus Minus Pre Read for the plus minus plate document then click Save Optional If you want to use this plate setup again you can save it as a template Select File gt Save As type a File Name then select Save As type sdt 4 Load the reaction plate into the instrument Note The A1 position is in the top left corner of the instrument tray Notes Chapter 4 Performing the Plus Minus Pre Read Run File View Tools Instrument Analysis Window Help Use 4R8BRBH rH ae Setup instrument YResuts y Instrument Control r Temperature PreRead Estimated Time Remaining hh mm Sample Heat Sink Cover Block Post Read z r Cycle _ Disconnect Status Stage Rep Time rnm ss Step State m Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Add Cy
17. Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown L o o mo Do mo Oe no no mo 0 u O i u o F Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown rn m Do Bc no Do no no no Ow 0 u u oO u u n Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown L mo mo Do Do Do no no no mao 0 oD i o u oD o u pm Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Do Dc m Dc Dc c Dc o Oe 0 o Notes Plus Minus Getting Started Guide for the 7300 7500 System 11 Chapter 1 Introduction Example Plus Minus Experiment Notes 12 Plus Minus Getting Started Guide for the 7300 7500 System Designing a Plus Minus Experiment Chapter 2 Workflow Designing a Plus Minus Experiment Using TaqMan probe based reagent configuration See page 14 Design the probe S 15 and primers icis Notes Plus Minus Getting Started Guide for the 7300 7500 System 13 A dssmusus Chapter 2 Designing a Plus Minus Experiment Y Using TagMan Probe Based Reagent Configuration Using TaqMan Probe Based Reagent Configuration About the Plus minus assays with an IPC use the fluorogenic 5 nuclease chemistry also known as Chemistry TaqMan probe based chemistry Chemistry TaqMan reagents or kits Description TaqMan probe based
18. by checking the Use box next to it e Select Task gt Unknown A U appears in the well next to the N 9 Select all remaining wells for the unknown samples IPC template and target template a Select wells B1 H12 by click dragging across all empty wells b Select your Target detector by checking the Use box next to it c Select Task gt Unknown A U appears in the boxes d Select the IPC detector by checking the Use box next to it e Select Task gt Unknown Another U appears next to the target U Notes New Document Wizard Setup Sample Plate Setup the sample plate with tasks quantities and detectors use ise E eor ueneno Te nin TAMRA NTC Unknown Standard NTC gt pets e wlale 4 A ET lt Dt lt ht ht spp Back Finish Cancel New Document Wizard Setup Sample Plate Setup the sample plate with tasks quantities and detectors Quencher Task Quantity Color Unknown Standard NTC Detector Reporter 1 2 3 4 5 6 UN 0D OD DA S ld Ed Ed Ed Ed mio lt Back Finish Cancel New Document Wizard Set Up Sample Plate Setup the sample plate with tasks quantities and detectors use Detector Reporter Quencher_ Task C VIC TAMRA Unknown Y Unknown Standard NTC 1 2 3 5 6 T 11 12 UJ OD QUE oA oA ug zm zm cu UE um EH ooo T 5m mp GR IE om Smp QD Q mD JOD OD OW mE JS qm 8 Qm Qm QD QD QUI Se QD QU C qm qom dmm dg qm
19. chemistry uses a fluorogenic probe to detect a specific PCR product as it accumulates during PCR cycles Process Polymerization FORWARD R REPORTER PRIMER 3 5 5 Y EN S S 2 e l l l lLI 5 Step 1 A reporter R and a quencher Q are attached to the 5 and 3 ends of a TaqMan Strand Displacement ao c won oe Oi Step 1 continued when both dyes are attached to the probe reporter dye emission is quenched probe Cleavage Polymerization Completed o amp m 5 3 o Pn 5 3 D 5 a won 5 3 5 Step 2 During each extension cycle the AmpliTaq Gold DNA polymerase cleaves the reporter dye from the probe Step 3 After being separated from the quencher the reporter dye emits its characteristic fluorescence Chemistry Kits for For more information about the TaqMan probe based chemistry refer to the Sequence Detection Systems Chemistry Guide PN 4348358 The following reagents are available from Applied Biosystems for designing and Plus Minus Assay running plus minus assays Part si Number TaqMan Exogenous Internal Positive Control Reagents with TaqMan 4308323 Universal PCR Master Mix TaqMan Universal PCR Master Mix 4304437 Note The IPC DNA primers and probe supplied in these reagents can be used with all sample target systems Refer to the TaqMan Universal PCR Master Mix Protocol PN 4304449 for instructio
20. iDa H amp ELE ES P Blew Results Instrument Control Temperature Estimated Time Remaining hh mm Sample Heat Sink Cover Block Post Head s Cycle Disconnect Status Stage Rep Time mm ss Step State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Add Cycle Add Hold Add Step Add Dissociation Stage Settings Sample Volume uL 50 v 9600 Emulation Amplification Plot Data Stage 1 Step 1 60 0 1 00 Help Ready Connected 74 Plus Minus Getting Started Guide for the 7300 7500 System Chapter 1 Introduction Example Plus Minus Experiment Amplify the DNA 1 Create an AQ plate document for amplification Follow the instructions as described in Chapter 5 Briefly in the New Document Wizard Select File gt New Select Absolute Quantification Standard Curve in the Assay drop down list In the Plate Name field type Plus Minus Amplification then click Next Note A standard curve is not needed for a non quantification amplification run Add detectors to the plate document see Appendix A then click Next Specify the detectors and tasks for each well then click Finish Type the sample names then save the document 2 Perform the plus minus amplification run a Select the Instrument tab b Select File gt Save As type a name for the Notes AQ
21. operator name and comments Hew Document Wizard a Select File gt New Assay Plus Minus b Select Plus Minus in the Assay drop down E BE cc urel Clear 2 list Template Blank Document c In the Plate Name field type Plus Minus Browse Pre Read then click Next Operator Administator SSS d Add detectors to the plate document see Comments Appendix A Creating Detectors then click Next e Specify detectors and tasks for each well then click Finish Rite Name Pus MmusPreRead f Double click each well to type the sample name then save the document Finish Tum 2 Enter the sample names and specify tasks in the Well Inspector View gt Well Inspector IMPORTANT If your experiment does not use all the wells on a plate do not omit the wells from use at this point You can omit unused wells after the run is completed For more information about omitting wells refer to the Online Help Notes 8 Plus Minus Getting Started Guide for the 7300 7500 System 3 Perform the plus minus pre read run a Select the Instrument tab By default the standard PCR conditions are displayed b Select File gt Save As type a name for the plus minus plate document then click Save Load the reaction plate into the instrument d Click Pre Read Notes Example Plus Minus Experiment Example Plus Minus Experiment Procedure File View Tools Instrument Analysis Window Help
22. refer to the Sequence Detection Systems Chemistry Guide PN 4348358 ARn vs Cycle Log This plot displays the Rn dye fluorescence as a function of cycle You can use this plot to identify and examine irregular amplification and to manually set the threshold and baseline parameters for the run setup Y j Instrument Y Results Y Fluorescence Plate Y Spectra Y Component Y Amplification Plot Y Standard Curve Y Component 9000 00 8000 00 7000 00 6000 00 6000 00 4000 00 3000 00 UT 2000 00 le A E He 0 00 Components Nl v ran Bo v Rox Bo c B v Tamra 7 PEATE ETT TD PL EVEL Cycles Set Y netument Yresuts V Plate Y Amplification Plot Y Standard Curve Rn Rn vs Cycle 2 12345 6 7 8 91011121314151617 18192021 22 2324 25 26 27 2829 3031 32 3334 35 36 37 38 38 40 Cycle Number R4 ABI Prism 7000 SDS Software E coli_amplification Absolute Quantification P E View Tools Instrument Analysis Window Help Jue Sa A B E e H Setup Y Instrument Plate Y Spectra Delta Rn Spectra Y Component Y Amplification Plot Y Standard Curve Delta Rn vs Cycle 1 0e 000 1 0e 001 1 0e 002 21 aig D f d Data Rin vs Cycle Detector E coli Line Color well Color Analysis Settings C Auto Ct Manual Ct Threshold 0 050860 Auto Baseline Manual Baseline Start cycle 3 End cycle 15 Analyze Data Delta An vs Cycle wv Detecto
23. 1 00 Ready Connected Help Save As Save in G SDS Documents gt ex E3 My Recent Documents Desktop My Documents l US File name Plus Minus Post Read aces SDS Documents sds Y Save as type Cancel A 40 Plus Minus Getting Started Guide for the 7300 7500 System Performing the Post Read Run 4 Load the reaction plate into the instrument Note The A1 position is in the top left Well A1 corner of the instrument tray 5 Click Post Read After the run is finished the status values and buttons are grayed out and a message indicates whether or not the run is successful 6 Click the green analysis button to start analysis All data generated during the run are saved to the plus minus plate document that you specified in step 3 Notes Plus Minus Getting Started Guide for the 7300 7500 System 41 Chapter 6 Performing the Plus Minus Post Read Run Performing the Post Read Run Example Experiment In the example plus minus experiment using an IPC the pre read run was subtracted from the post read run to account for background fluorescence Post read results for the presence of E coli are displayed in the Results gt Plate tab For an explanation of results see Viewing Plus Minus Results on page 43 No template n Mo template n No template n No template n No template n
24. 31 32 33 3435 36 37 38 39 40 Cycle Number Example Experiment The image below shows the amplification plot for unknown sample well G2 which displays a negative result The sample is negative because the amplification for the E coli target sequence is less than the NAC signal The IPC amplification demonstrates that PCR was successful for this well Setup Y instrument Results Plate Y Spectra Y Component Y Amplification Plot Y Standard Curve Delta Rn vs Cycle 0 28 G Unknown Data Delta Rn vs Cycle Detector fan 0 24 Line Color Detector Color 0 20 Analysis Settings Auto Ct 0 16 C Manual Ct Threshold mixed IPC Well D7 E 0 12 Auto Baselin E T 0 08 Start cycle amp uto End cycle amp uto 0 04 p No E coli present 0 0 04 123458728238101112131415161718192021222324252B 27 2829 30 31 32 333435 35 37 38 38 40 Cycle Number Notes Plus Minus Getting Started Guide for the 7300 7500 System 45 Chapter 6 Performing the Plus Minus Post Read Run Viewing Plus Minus Results Example Experiment The image below shows the amplification plot for unknown sample well F1 which displays a positive 4 result The sample is positive for the E coli target sequence because it has amplification above the IPC threshold Setup Y Instrument Y Resutts Y Plate Y Spectra Y Component Y Amplification Plot Delta Rn vs Cycle 070
25. Minus Getting Started Guide for the 7300 7500 System 47 Chapter 6 Performing the Plus Minus Post Read Run Exporting Plus Minus Plate Data Notes 48 Plus Minus Getting Started Guide for the 7300 7500 System Creating Detectors Before you can use a plate document to run a plate create and apply detectors for all samples on the plate A detector is a virtual representation of a gene or allele specific nucleic acid probe reagent used for analyses performed on instruments To create a detector 1 Select Tools Detector Manager Note A plate document any type must be open before you can access the Tools menu 2 Select File New 3 Enter a name for the detector IMPORTANT The name of the detector must be unique and should reflect the target locus of the assay such as IPC or E coli Do not use the same name for multiple detectors 4 Optionally click the Description field then enter a brief description of the detector Notes Detector Manager Detector List Find al Lost Mod IPC VIC TAMRA 20034 0 2 18 HTR4 FAM none 2003 09 15 08 GTF2B FAM none 2003 09 15 08 GTF21 FAM none 2003 09 15 08 GAPDH VIC FAM none 2003 09 15 08 GAPDH FAM none 2003 09 15 08 none 2003 09 15 08 2003 09 15 08 2003 0 12 16 2003 09 15 08 2003 09 15 08 2003 09 45 08 onnamans na Y none none none none fname Add To Plate Document H
26. PN 4322547 and protocol PN 4318925 to obtain a final concentration of 10 ng uL of DNA for each sample Notes 18 Plus Minus Getting Started Guide for the 7300 7500 System Setting Up the Reaction Plate Setting Up the Reaction Plate This section describes how to set up a 96 well plate for a plus minus run with samples and reaction mix The reagents volumes and final concentrations in Preparing the PCR Reaction Mix on page 20 were taken from the TaqMan Exogenous Internal Positive Control Reagents Protocol PN 4308323 Example Experiment Extracted DNA samples are pipetted onto a 96 well plate along with negative and positive controls Wells A1 A6 contained blocked IPC and no target template wells A7 A12 contained IPC template IPC but no target template and wells B1 H12 contained both IPC and target template Contains blocked IPC and no target 5 6 7 8 9 10 11 12 NAC NTC plus IPC Contains IPC and no target Samples Unknown plus IPC Contains IPC plus target X GG mnm mgogo O UU gt GR2363 Notes Plus Minus Getting Started Guide for the 7300 7500 System 19 Chapter 3 Setting Up the Reaction Plate Setting Up the Reaction Plate Preparing the PCR Reaction Mix 1 Make a volume of reaction mix sufficient to provide 45 uL for each well you use on the plate Volimso ope Final
27. Performing the Amplification Run Performing the Amplification Run 1 Select the Instrument tab By default the standard PCR conditions for the File View Tools Instrument Analysis Window Help Uae e EE E H e 1 a Results Y Instrument Control Temperature PCR re displ C step are d Sp ayed Estimated Time Remaining hh mm Sample Heat Sink Cover Block Cycle Discont Status Stage Rep Time mm ss Step ena State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 Add Cycle Add Hold Add Step Add Dissociation Stage Help r Settings Sample Volume pL 50 Iv 9600 Emulation Amplification Plot Data Stage 3 Step 2 60 0 1 00 v Ready Connected UM Times and Temperatures Initial Steps PCR Each of 40 cycles AmpErase UNG Activation AmpliTaq Gold DNA Melt Anneal Extend Polymerase Activation HOLD HOLD CYCLE CYCLE 2 min 50 C 10 min 95 C 15 sec 95 C 1 min 60 C 2 Accept default values for e Sample volume 50 uL e 9600 Emulation selected Note The 9600 Emulation feature is not available for the 7300 instrument Notes 36 Plus Minus Getting Started Guide for the 7300 7500 System 3 Select File gt Save then click Save to retain the name you assigned when you created the plate document 4 Load the reaction pla
28. Plate document then click Save Load the reaction plate into the instrument then click Start By default the standard PCR conditions for the PCR step are displayed After the run a message indicates if the run is successful or if errors were encountered Hew Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Absolute Quantification Standard Curve Container 96 Well Clear Template Blank Document Browse Operator amp dministrator Comments EN ame Plus Minus Amplification File view Tools Instrument Analysis Window Help 48H amp HH Instrument Control Temperature Estimated Time Remaining hh mm Sample Heat Sink Cover Block Cycle Status Stage Time mm ss State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 Add Cycle Add Hold Add Step Add Dissociation Stage Help Settings Sample Volume pL 50 Iv 9600 Emulation Amplification Plot Data Stage 3 Step 2 60 0 1 00 Y Connected 10 Plus Minus Getting Started Guide for the 7300 7500 System Example Plus Minus Experiment Example Plus Minus Experiment Procedure Perform the plus minus post read run 1 7 Open the plus minus pre read plate document File View Tools Instrument Analysis Window Help n E 9 then use it to p
29. Plus Minus Assay Applied Biosystems 7300 7500 Real Time PCR System Unknown Unknown Unknown Unknown Unknown Unknown Unknown gt Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown mn FE d D 7500 Real Time PCR System Introduction Designing a Plus Minus Experiment Setting Up the Reaction Performing a Plus Minus Pre Read Run Generating Amplification Data Performing a Plus Minus Post Read Run Copyright 2004 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Authorized Thermal Cycler This instrument Serial No is an Authorized Thermal Cycler Its purchase price includes the up front fee component of a license under United States Patent Nos 4 683 195 4 683 202 and 4 965 188 owned by Roche Molecular Systems Inc and under corresponding claims in patents outside the United States owned by F Hoffmann La Roche Ltd covering the Polymerase Chain Reaction PCR process to practice the PCR process for internal research and development using this instrument The running royalty component of that license may be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents This instrument is also an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems Its use with Auth
30. Select File gt Open gt Spot Set Two user attention words appear in this Applied Biosystems user documentation Each word 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 chemistry kit use or safe use of a chemical Refer to the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Getting Started Guide PN 4347828 and the Applied Biosystems 7300 7500 Real Time PCR System Site Preparation Guide PN 4347823 for important safety information Plus Minus Getting Started Guide for the 7300 7500 System Vil Preface How to Obtain More Information How to Obtain More Information For more information about using the 7300 7500 system refer to Applied Biosystems 7300 7500 Real Time PCR System Online Help e Applied Biosystems 7300 7500 Real Time PCR System Allelic Discrimination Getting Started Guide PN 4347822 Applied Biosystems 7300 7500 Real Time PCR System Absolute Quantification Getting Started Guide PN 4347825 Applied Biosystems 7300 7500 Real Time PCR System Relative Quantification Getting Started Guide PN 4347824 Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Getting Started Guide PN 4347828 e Applied Biosystems 7300 7500 Real Ti
31. and plant tissue e PrepMan Ultra Sample Preparation Reagent Kit e Applied Biosystems PN 6100 01 e Applied Biosystems PN 4346860 e Applied Biosystems PN 4340274 e Applied Biosystems PN 4322547 Labeled primers and probes source e Primer Express Software custom designed primers and probes e PN 4330710 1 user license PN 4330709 10 user license PN 4330708 50 user license MicroAmp Optical 96 Well Reaction Plates Applied Biosystems PN 4306757 Optical Adhesive Covers Applied Biosystems PN 4311971 Reagent tubes with caps 10 mL TaqMan Exogenous Internal Positive Control Reagents VIC Probe Applied Biosystems PN 4305932 Applied Biosystems PN 4308323 TaqMan Universal PCR Master Mix Applied Biosystems PN 4304437 Centrifuge with adapter for 96 well plates Major Laboratory Supplier MLS Gloves MLS Microcentrifuge MLS Microcentrifuge tubes sterile 1 5 mL MLS Nuclease free water MLS Pipette tips with filter plugs MLS Pipettors positive displacement MLS Tris EDTA TE Buffer pH 8 0 MLS Vortexer MLS Plus Minus Getting Started Guide for the 7300 7500 System Chapter 1 Introduction Example Plus Minus Experiment Example Plus Minus Experiment Notes Overview Description To better illustrate how to design perform and analyze plus minus experiments this section provides an example expe
32. and quencher information and optionally the gene name or symbol for the sample name You can view the contents in a spreadsheet program such as Microsoft Excel Notes 50 Plus Minus Getting Started Guide for the 7300 7500 System Viewing Amplification Data Specifying Analysis Settings Before you analyze specify parameters to enable auto baseline and auto threshold calculations Unless you have already determined the optimal baseline and threshold settings for your experiment you need to analyze data twice first using the automatic baseline and threshold feature of the SDS software Auto Cy and again after determining the optimal baseline and threshold for your data To specify analysis settings 1 Click x or select Analysis gt Analysis Settings 2 In the Detector drop down list select AIl 3 Select Auto Ct to set the SDS software to automatically generate baseline and threshold values for each well in the study IMPORTANT After analysis you must verify that the baseline and threshold were called correctly for each well Alternatively you can select Manual Ct and specify the threshold and baseline manually For more information about manually adjusting C refer to the Online Help 4 Select Use System Calibration to use the calibration files Background and Pure Dye that are stored on the computer rather than the calibration information that is stored in the plate document itself For
33. ces that make this new Integrated Science possible 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 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 www appliedbiosystems com Applied Bibbvstems Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 01 2004 Part Number 4347821 Rev A an Applera business
34. cle Add Hold Add Step Settings Sample Volume uL 50 Iv 9600 Emulation Amplification Plot Data Stage 1 Step 1 60 0 1 00 Help Ready Connected 74 Save in SDS Documents j ex EJ My Recent Documents My Documents Plus Minus Pre Read gt sps Documents sds Cancel 2 My Network Places File name Save as type Keyed corner 28 Plus Minus Getting Started Guide for the 7300 7500 System Performing the Pre Read Run 5 Click Pre Read File View Tools Instrument Analysis Window Help O 9 During the pre read run the instrument collects d one fluorescence Scan per well Instrument Control Temperature Estimated Time Remaining hh mm Sample Heat Sink As the instrument performs the run it displays PostHead Cavar Block Cycl status information in the Instrument tab After Disconnect Status Bre Rep Idi Ak A the run is finished the status values and the nin ae PY buttons are greyed out and a message indicates whether or not the run is successful 6 Select File gt Close Notes Plus Minus Getting Started Guide for the 7300 7500 System 29 Chapter 4 Performing the Plus Minus Pre Read Run Performing the Pre Read Run Notes 30 Plus Minus Getting Started Guide for the 7300 7500 System Chapter 5 Generating Amplification Data Workflow
35. d IPC Notes 20 Plus Minus Getting Started Guide for the 7300 7500 System A No enna No fenplafeotemolte Votempisre Ko erate No Template Na Terolate Motempisie hn feng Nn temple Nr Teme Tlotempiee E mu m u um Su m mJ mum 8 Gg HH m o u D D ul D a D urinown Unknown Urkawr Unovn Unknown Unknown Unknown Unknown Unknown Unknown known Unenawn 7 Performing the Plus Minus iiss hapter a ARERA Pre Read Run 5 m nown unknown urkrawn unknown Lnimewn Unknown Urkrowr Unknown Lnnewn Unknown Unknown unknown Workflow Performing the Plus Minus Pre Read Run Create a Plus Minus See page 22 plate document Perform the pre read run See page 28 Notes Plus Minus Getting Started Guide for the 7300 7500 System 21 Chapter 4 Performing the Plus Minus Pre Read Run The Pre Read Run The Pre Read Run A pre read run records the background fluorescence of each well of the plus minus plate before PCR During the post read run the pre read fluorescence 1s subtracted from the post read fluorescence to account for pre amplification background fluorescence which ensures more accurate results Before You Begin Check that background and pure dye runs have been performed regularly to ensure optimal performance of the 7300 7500 system For more information about calibrating the 7300 7500 system refer to the Online Help Creating a Plus Minus Plate Document Plate Docume
36. eline fluorescence determined during the pre read run then assigns positive or negative calls using the amplified data Plus Minus Getting Started Guide for the 7300 7500 System Example Plus Minus Experiment Example Plus Minus Experiment Procedure Example Plus Minus Experiment Procedure Design the experiment and prepare DNA 1 Design the experiment as explained in Chapter 2 a Order the TaqMan Exogenous Internal Positive Control Reagents kit and the TaqMan Universal PCR Master Mix b Design the primers and FAM labeled probe set for E coli detection with Applied Biosystems Primer Express software 2 Extract DNA from samples see Preparing DNA on page 18 using the PrepMan Ultra sample Preparation Reagent Kit PN 4322547 and protocol PN 4318925 to obtain a final concentration of 10 ng uL of DNA for each sample 3 Prepare sufficient reaction mix see Preparing i E item Volume for one Final the PCR Reaction Mix on page 20 by using the Reaction Concentration volumes as listed in the table on the right TaqMan 25 0 1X o Universal PCR Master Mix 2X Nnm CHEMICAL HAZARD c Lu ad TaqMan Universal PCR Master Mix may e a IPC Mix 5 0 50 to 900 nM cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read 90x Exo IPC 1 0 50 to 900 nM the MSDS and follow the handling instructions ro Wear appropriate protective eyewear clothing Target primer
37. elp Duplicate Done Addto Plate Document Import Export Clear Clear All Properties Hew Detector M ame Description 4 Reporter Dye Quencher Dye Color Mates Create Another Cancel Plus Minus Getting Started Guide for the 7300 7500 System 49 Appendix A 5 In the Reporter Dye and Quencher Dye drop down lists select the appropriate dyes for the detector Note The dyes that appear in the Reporter and Quencher Dye lists are those that have been previously entered using the Dye Manager If the dye that you want to use does not appear in a list use the Dye Manager to add the dye and then return to step 5 in this procedure Refer to the Online Help for more information 6 Click the Color box select a color to represent the detector using the Color dialog box then click OK f Optionally click the Notes field then enter any additional comments for the detector 8 Click OK to save the detector and return to the Detector Manager 9 Repeat steps 2 to 8 for the remaining detectors 10 In the Detector Manager click Done when you finish adding detectors Example Experiment In the example plus minus experiment a detector was created for the E coli target and another was created for the IPC The E coli detector was assigned a blue color and IPC a black color Note When creating detectors you use the reporter dye
38. erform the post read run See EE aes SENAD Performing the Post Read Run on page 40 Instument Conta Temperature Pre Read Estimated Time Remaining hh mm Sample Heat Sink Cover Block a Select the Instrument tab a Disconnect Status Stage Rep b Select File gt Save As type a name for the G m plus minus post read plate document then Ne mam click Save Thermal Profile Auto Increment Ramp Rate Load the reaction plate into the instrument d Click Post Read Add Cycle Add Hold Add Step Help Settings Sample Volume pL 50 Amplification Plot Data Stage 1 Step 1 60 0 1 00 Y Ready e Click W or select Analysis gt Analyze j Click the Results tab to view results for mu ccGK S No template n No template n No template n Mo template n No template n No template n No template No template No template each well m m D m m N N 2 If you need to troubleshoot the plus minus Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown L eee aa T Mc Ic Ic Io Io Do Do E o 0 results see Viewing Amplification Data on i il i o E D I page 51 C Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown L mo mo mo mo Do no no no mo 0 m u u u u O u o D Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown rn m H mo mo mo no no Oe Ow 0 u u u o Oo E
39. es a signal that indicates specific amplification Normalized reporter The ratio of the fluorescence intensity of the reporter dye signal to Rp the fluorescence intensity of the passive reference dye signal Delta R AR The magnitude of the signal generated by a set of PCR conditions AR R baseline The figure below is a representative DNA amplification plot and includes some of the terms defined above Threshold l No Template Control Baseline C 7 0 5 10 15 20 25 30 35 4 Cycle Number o Starting the To analyze the amplification data AQ plate click y or select Analysis gt Analyze Analysis The software generates several types of result views as described in the following section Notes 52 Plus Minus Getting Started Guide for the 7300 7500 System Viewing the Amplification Data About the Results Tab Viewing the Amplification Data About the Results Tab In the Results tab you can view the results of the amplification run and change the parameters to run the plate document again or reanalyze the data The Results tab has seven secondary tabs Details about each tab are provided in the Online Help Plate Tab Displays the results data of each well including The sample name and detector task and color for each well A calculated value quantity default displays are not determined for runs without standard curves ARn or Cy Select Analysis g
40. ess requires a license For information on obtaining licenses contact the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or The Licensing Department Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 USA Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document TRADEMARKS Applied Biosystems ROX VIC TAMRA MicroAmp and Primer Express are registered trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries AB Design Applera ABI PRISM BloodPrep Celera Genomics FAM iScience iScience Design NucPrep and PrepMan are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries TaqMan is a registered trademark of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Part Number 4347821 Rev A 1 2004 Plus Minus Getting Started Guide for the 7300 7500 System About the About Plus Minus About Plus Minus 7300 7500 system assays using IPC assays Chapter 1 aia Introduction
41. he plus minus Pe tssesrs YRerc 7S KS ESE calls for the presence or absence of the target A gg m template n H tennis Midi emple template T template 4 sequence for each well u a The following Example Experiment boxes show the B Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Hc Hc Mc S e o e Ic mo amplification plots of each type of result NAC NTC n o o Hn HH 0 0 plus minus and undetermined C Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown c D o Ow Ow Ow o Ute m o LU m Note The amplification plots are obtained from the AQ plate document used to amplify the samples in D Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown the 96 well plate Do n no nno nno no o Do oO 1 1 E Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown nno nno nno nno nno nno nno nno m u F Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown nno nno no nno nno nno nno nno i Ui G Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown ute Oc Ow c ute Dco Dco Dco i Example Experiment The image below displays the amplification plot for well A1 the No Amplification Control NAC which contains blocked IPC and no target template This plot demonstrates that there is no amplification for IPC o
42. ion Prepare DNA See page 18 Set up reaction plate See page 19 Notes Plus Minus Getting Started Guide for the 7300 7500 System 17 Chapter 3 Setting Up the Reaction Plate Preparing DNA Preparing DNA Systems and Applied Biosystems supplies several instrument systems and chemistries for isolating Chemistries for DNA from a variety of starting materials such as blood tissue cell cultures plant DNA Isolation material and food System Part Number BloodPrep Chemistry 4346860 NucPrep Chemistry 4340274 PrepMan Ultra Sample Preparation Reagent Kit 4322547 ABI Prism 6100 Nucleic Acid PrepStation 6100 01 For more information refer to e DNA Isolation from Fresh and Frozen Blood Tissue Culture Cells and Buccal Swabs Protocol PN 4343586 e NucPrep M Chemistry Isolation of Genomic DNA from Animal and Plant Tissue Protocol PN 4333959 Quality of DNA Ensure that the DNA you use for a plus minus experiment e Has a A sonsgo ratio of gt 1 7 s extracted from the raw material you are testing using an optimized protocol Does not contain PCR inhibitors e s intact as visualized by gel electrophoresis Has not been heated above 60 C which can cause degradation Example Experiment The meat samples are frozen with liquid nitrogen and ground to a fine powder with a pre chilled mortar and pestle DNA is extracted using the PrepMan Ultra Sample Preparation Reagent Kit
43. ion About the 7300 7500 System About the 7300 7500 System Description Plus Minus Assay The Applied Biosystems Real Time PCR System 7300 7500 system uses fluorescent based PCR chemistries to provide Quantitative detection of nucleic acid sequence using real time analysis Qualitative detection of nucleic acid sequence using end point and dissociation curve analysis The 7300 7500 system allows you to perform several assay types with plates or tubes in the 96 well format This guide describes the plus minus assay which determines whether or not a specific target sequence is present in a sample Note For information about the other assay types refer to the Sequence Detection Systems Chemistry Guide PN 4348358 and the Online Help for the 7300 7500 system About Plus Minus Assays Using an IPC Definition What Is An IPC Notes A plus minus assay is an end point assay that determines if a specific target sequence 1s present plus or not present minus in a sample In an end point assay data are collected at the end of the PCR process An IPC is an internal positive control see TaqMan Exogenous Internal Positive Control Reagents kit PN 4308323 that 1s used in plus minus assays to monitor the PCR process and to ensure that a negative result 1s not due to failed PCR The IPC consists of a template a primer set and a dye labeled VIC probe that are added to each well of a reaction plate the IPC is part
44. mation on each well in the Setup tab Creating a Plus Minus Plate Document Creating a New Plus Minus Plate Document well Inspector Wells A1 46 Sample Name NAC use Detector Reporter v Pc TAMRA NTC v E coli FAM E Omit Well Add Detector Remove Close Passive Reference ROX Example Experiment and the Ul box seen below is the unknown sample plus IPC JE Lm Hac NAT 3 rrean Lire raai C Urarcren rizen Liring m 7 n n B iriri Lrknownm ie i d g n F E l Liro Ui iire ie Urar Wij i ii Im i Ba reser LirienenveTs LP veri Lire eet rarei E o o oO o G Urrira Ea riie Lirdcracreem Lina rifia Lira facem ul m u u u I i oO n t oO oO H Unifi Likra Liira Liria Lraracesem Lira races jut Uu J HAC Nat NTC NTC NIC NTC i i i D a Lidice Lirie Uninet Urry n Leet Lire Er rerenm Urrira u m m u oO o o oO Lien Lrarcreen riria Lirkricreer Lrkriceern Ur rkeen oO nice rice a Lir Lirdancrssei s E E ER ER Linken Lib FERAT gra m o Lr nown LDrn noen Uningan Unie Unon U rknewn m m Liki Unman UnA m a oO oO Lirriceaem Faiga ij Unkriceesm BS The pre read plate document we created with controls and samples is shown in the picture below We selected two detector tasks for each well one for the target and one for the IPC
45. me PCR System Site Preparation Guide PN 4347823 e Sequence Detection Systems Chemistry Guide PN 4348358 How to Obtain Services and 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 page you can e Search through frequently asked questions FAQs e Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Services and Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com viii Plus Minus Getting Started Guide for the 7300 7500 System Chapter 1 Overview Introduction Notes Introduction About the S 2 7300 7500 system oe About Plus Minus assays See page 2 using an IPC ee About Plus Minus See page 4 assays e Example Plus Minus experiment or Plus Minus Getting Started Guide for the 7300 7500 System Chapter 1 Introduct
46. more information about system calibration files refer to the Online Help 5 Click OK amp Reanalyze Notes Analysis Settings Absolute Quantification Ct Analysis Detector All l f Auto Ct Manual Ct Threshold 0 728583 F Start cycle Auto End cycle Auto Use System Calibration f OK amp Reanalyze Cancel Apply Online Help E File view Tools Instrument Analysis Window zn j Ll Ed Ej zz b E P Contents and Index J Setup Y Instrument Y Results V Plus Minus Getting Started Guide for the 7300 7500 System 51 Appendix B Analyzing the Plus Minus Amplification Data AQ Plate Analyzing the Plus Minus Amplification Data AQ Plate Terms Used in The following are terms commonly used in quantification analysis Quantification Analysis Term Definition Baseline A line fit to fluorescence intensity values during the initial cycles of PCR in which there is little change in fluorescence signal Threshold cycle Cy The fractional cycle number at which the fluorescence intensity exceeds the threshold intensity Passive reference A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescence fluctuations caused by changes in concentration or volume Reporter dye The dye attached to the 5 end of a TagMan probe The dye provid
47. name and comments Assay Absolute Quantification Standard Curve Container 95 well Clear ba Template Blank Document Browse Operator Administrator Comments En eg _ Plus Minus Amplification New Document Wizard Select Detectors Select the detectors you will be using in the document Passive Reference ROX Detector Hame Description Reporter Quenche Detectors in Document Internal Positiv IC none none Ex Remove none Remove none none none none none none f nane lt New Detector lt Back Next gt Finish Cancel Plus Minus Getting Started Guide for the 7300 7500 System 33 Chapter 5 Generating Amplification Data Creating a Plate Document for Sample Amplification f Select six wells on the plate document for the no amplification control blocked IPC and no target template a Select wells A1 A6 b Select the target detector by checking the Use box next to it c Select Task gt NTC An N appears in the well d Select the IPC detector by checking the Use box next to it e Select Task gt NTC A second N appears in the well 8 Select six more wells on the plate document for the no template control IPC but no target template a Select wells A7 A12 b Select the target detector by checking the Use box next to it c Select Task gt NTC An N appears in the well d Select the IPC detector
48. nd Ad No template no IPC E coli NTC 0 138 Std Dev C 1 P NTC 1 417 T AS Ma template na IPC E coli NTC 0 133 P NTC 1 357 You can format the display of the report and how the AB No template no IPC Ecol NTC 0132 report is printed Refer to the Online Help for more Mes Pee pon information ie PE 19 B2 Unknown E coli Unknown 0 759 P P 1 199 B3 Unknown E coli Unknown o 744 IFS IFS 1 126 B4 Unknown E coli Unknown J 0 133 IFS IFS 1 731 Adjusting Graph Settings Graph Settings Clicking on the Spectra Component Amplification Real Time Settings Post Run Settings Plot and Standard Curve displays the Graph Settings ied ag m Minimums NE z SCC dialog box which allows you to adjust the plot s s eoe TG SN settings Masimum 10 Mes Axle Is autascaled in HealTime W Auto Scale The adjustable settings depend on which plot you are viewing Refer to the Online Help for more information Minimum Masimum Display Options Line Width 2 Defaults Cancel Apply Notes Plus Minus Getting Started Guide for the 7300 7500 System 55 Appendix B Viewing the Amplification Data Overview of Result Calls Go to the plate Results tab to view the plus and minus calls for the presence or absence of the target sequence for each well For further determination of the results check the plots for each well Notes Setup Y Instrument Y Results Plate Y Spectra Y Report Y T Ee DERE ER ERR ENT
49. nown Dc Unknown Dc Unknown Oo Sr 56 Plus Minus Getting Started Guide for the 7300 7500 System References Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods Enzymol 155 335 350 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 Plus Minus Getting Started Guide for the 7300 7500 System 57 08 Plus Minus Getting Started Guide for the 7300 7500 System Numerics 7300 7500 system background and pure dye runs 22 description 2 plus minus assay 2 9600 Emulation 28 36 96 well reaction plate 7 19 loading 28 A absolute quantification 10 amplification data analyzing 51 viewing 53 amplification plates detectors creating 49 starting a run 37 amplification plot 44 45 Amplification Plot tab 54 amplification run PCR conditions 36 performing 36 Analysis button 37 analysis settings 51 Applied Biosystems contacting vili customer feedback on documentation viii Services and Support viii Technical Communications viii Technical Support viii auto Ct 51 B baseline 51 52 C calibration background and pure dye 51 Color box 50 Component tab 54 conven
50. ns on optimizing amplification of your target Notes 14 Plus Minus Getting Started Guide for the 7300 7500 System Designing the Probe and Primers Chemistry Kits for Plus Minus Assay Designing the Probe and Primers Design a probe and primer set for your target sequence Applied Biosystems provides the Primer Express software for this purpose For more information about using this software refer to the Primer Express Software v2 0 User Manual PN 4329500 Example Experiment In the example experiment we extracted DNA from 84 batches of hamburger meat and tested them for the presence of E coli using the plus minus assay on the 7300 7500 Real Time PCR System Six no IPC no target template controls six IPC no target template controls and 84 unknown samples were run For the example experiment the TaqMan Exogenous Internal Positive Control Reagents Kit supplies one 1 mL tube of 105 Exo IPC Mix This mix contains the IPC primers and VIC labeled probe The primers probe set for E coli was designed by Applied Biosystems Primer Express software and contained a FAM labeled probe with TAMRA as the quencher Notes Plus Minus Getting Started Guide for the 7300 7500 System 15 Chapter 2 Designing a Plus Minus Experiment Designing the Probe and Primers Notes 16 Plus Minus Getting Started Guide for the 7300 7500 System oetting Up the Reaction Plate Workflow Setting Up the React
51. nt Parameters Notes A plus minus plate document is an SDS software document that stores data collected from a plus minus run for a single 96 well plate Plus Minus plate documents also store other information about the run including sample names and detectors When you create a plus minus plate document with an IPC you define specific parameters for each plus minus reaction plate Detectors A virtual representation in the SDS software of a TaqMan probe and primer set and an associated fluorescent dye that detects a single target nucleic acid sequence Appendix A explains how to create detectors e Task A setting that you apply to each well of a plate document that determines the way the SDS software uses the data collected from the well during analysis Note Applied Biosystems recommends you run six replicates of each control NAC and NTC to accurately define plus minus thresholds and obtain plus minus calls with a 99 7 confidence level 22 Plus Minus Getting Started Guide for the 7300 7500 System Creating a Plus Minus Plate Document Detector Tasks You assign a task to each detector in each well of a plate document For plus minus plate documents there are four types of tasks Task Symbol Apply to Unknown m All detectors of wells that contain target sequence IPC D All detectors of wells that contain IPC IPC All detectors of control wells that contain IPC but no ta
52. of the reaction mix see Preparing the PCR Reaction Mix on page 20 Plus Minus assays with an IPC use fluorogenic 5 nuclease chemistry also known as TaqMan probe based chemistry During amplification the sample target and the IPC target generate reporter fluorescence signals such that positive or negative calls may be made on unknown samples Note The SYBR Green I dye chemistry is not supported for plus minus assays using an IPC Plus Minus Getting Started Guide for the 7300 7500 System About Plus Minus Assays Using an IPC Terms Used in Plus Minus Analysis Terms Used in Plus Minus Term Definition Analysis Internal positive control IPC A second TaqMan probe and primer set added to the reaction plate to monitor the PCR process and to ensure that a negative result is not due to failed PCR in the sample No amplification control NAC Wells that contain no target template and blocked IPC the IPC target template has been blocked by a blocking agent No template control NTC A sample that contains no target template Nucleic acid target Nucleotide sequence that you want to identify as present or absent Unknown sample U The sample for which you want to determine the presence or absence of a specific target Notes Plus Minus Getting Started Guide for the 7300 7500 System 3 Chapter 1 Introduction About Plus Minus Assays About Plus Minus Assays
53. orized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents Purchase of this product does not itself convey to the purchaser a complete license or right to perform the PCR process Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 DISCLAIMER OF LICENSE No rights for any application including any in vitro diagnostic application are conveyed expressly by implication or by estoppel under any patent or patent applications claiming homogeneous or real time detection methods including patents covering such methods used in conjunction with the PCR process or other amplification processes The 5 nuclease detection assay and certain other homogeneous or real time amplification and detection methods are covered by United States Patent Nos 5 210 015 5 487 972 5 804 375 and 5 994 056 owned by Roche Molecular Systems Inc by corresponding patents and patent applications outside the United States owned by F Hoffmann La Roche Ltd and by United States Patent Nos 5 538 848 and 6 030 787 and corresponding patents and patent applications outside the United States owned by Applera Corporation Purchase of this instrument conveys no license or right under the foregoing patents Use of these and other patented processes in conjunction with the PCR proc
54. ple plus minus experiment 6 sample plus minus experiment procedure 7 Sequence Detection Systems Chemistry Guide 2 14 Services and Support obtaining viii Setup tab 27 software 22 Spectra tab 53 starting an amplification plate run 37 T TAMRA quencher 15 TaqMan Exogenous Internal Positive Control Reagents Kit 7 14 TaqMan probe based chemistry 14 TaqMan Universal PCR Master Mix 6 7 14 19 protocol 14 target template 3 25 task 22 Technical Communications contacting vili Technical Support contacting viii template documents 27 text conventions vii threshold 51 threshold cycle auto Ct 51 definition 52 Tools menu 49 Training obtaining information about viii troubleshooting 11 32 U unknown sample 3 26 unknowns 20 Use checkbox 34 Use System Calibration checkbox 51 V VIC labeled probe 15 Plus Minus Getting Started Guide for the 7300 7500 System W Well Inspector window 8 26 workflow plus minus post read 39 plus minus pre read run 21 plus minus reaction plate 17 61 62 Plus Minus Getting Started Guide for the 7300 7500 System Science iScience To better understand the complex interaction of biological systems life scientists are developing revolutionary approaches to discovery that unite technology informatics and traditional laboratory research In partnership with our customers Applied Biosystems provides the innovative products services and knowledge resour
55. qq gm qmm qmm co JS JS 6 JE ES ES Se Se qom Qo JOD UST JSR JS JS JS gp J ee ee TNT he he he Pe ee ee qm qom qom qom jo lt Back Finish Cancel 34 Plus Minus Getting Started Guide for the 7300 7500 System Creating a Plate Document for Sample Amplification 10 Click Finish The 7300 7500 SDS software creates the plate document 11 Enter sample names for each well a Double click one well to open the Well Inspector or select View gt Well Inspector b Click drag to select all replicate wells for that sample c Type the sample name in the Well Inspector The information appears in the selected 7 me Use Detector Reporter Quencher Task Color well s v PC VIC TAMRA NTC iE V E coli FAM TAMRA INTC d Accept the default setting ROX for the Passive Reference ROX dye Optionally you can change the detector task and Passive Reference dye e Repeat steps b through d until all wells have names f Click 4 to close the Well Inspector IMPORTANT If your experiment does not use all the wells on a plate do not omit the wells from use at this point You can omit unused wells after the run For information about omitting unused wells refer to the Online Help g Verify the information about each well in the Setup tab Notes Plus Minus Getting Started Guide for the 7300 7500 System 35 Chapter 5 Generating Amplification Data
56. r E coli X Line Color well Color Analysis Settings Auto Ct C Manual Ct hreshold o1 13867 93 J uy Start cycle Auto End cycle Auto 1 0e 004 Analyze 1 0e 005 Notes 54 Plus Minus Getting Started Guide for the 7300 7500 System Viewing the Amplification Data Report Tab Ct VS Well Position Plot B File View Togs Instrument Analysis Window Help Jie amp l 88H eA Setup Instrument This plot displays threshold cycle C4 as a function ponen areacaton Pot anter Cune ftesocten Y epar of well position You can use this plot to locate Ct vs Well Position outliers from detector data sets Data Ct vs Well Position w Detector E coli m Line Color Analysis Settings 7 Auto Ct S na u mamm Pee eee Magun ee m Mapan go Threshold 0 113867 G o f Start cycle Auto End cycle amp uto Analyze 0 1 4 7 1013161922252831 3437 40 43 46 49 52 555861 6467 707376 7982858891 94 Well Position Report Tab Setup Instrument Y Results Plate Y Spectra Y Report This tab displays data for selected wells in a table well SampleName Detector Task Call Rn format The data columns associated with the report pu CE c E ER are determined by the assay type For plus minus CN s ool Mu JE assays the following data columns are available A3 Notemplate no IPC E coli NTC 0148 IFS NTC 1 385 Well sample Name Detector Task Cy a
57. r the E coli target sequence 7 Setup 7 Instrument Y Results i SS S S ll 6 uQS Q Q Q Plate Amplification Plot Standard Curve Delta Rn vs Cycle 0 01 Data Delta An vs Cycle v Detector All Line Color Detector Color 0 00 Analysis Settings Auto Ct C Manual Ct Well A1 DIE nacer No E coli present 0 00 c vIn E a a a Start cycle Auto igs Em Blocked IPC no amplification End cycle amp uto 0 00 0 0 12345 6 7 8 9 10111213141516171819 20 21 2223 24 25 26 27 2829 30 31 32 3334 35 36 37 38 39 40 Cycle Number Notes 44 Plus Minus Getting Started Guide for the 7300 7500 System Viewing Plus Minus Results The Plate Tab Example Experiment The image below shows the amplification plot for well A10 the No Template Control NTC which contains IPC but no target template This plot demonstrates a positive amplification curve for IPC and no amplification for the E coli target sequence J Plate Y Spectra Y Component Y Amplification Plot V Standard Curve Delta Rn vs Cycle 0 06 Data Delta Rn vs Cycle Detector A Line Color Detector Color v 0 05 Analysis Settings 0 04 Auto Ct C M I Ct anua IPC 0 03 Threshold mixed c a G line sx a C M 0 02 Start cycle amp uto 0 01 End cycle Auto al No E coli present 1 1234 5 6 7 8 9 10111213141516171819 2021 2223 24 25 26 27 2829 30
58. rget template NTC M All detectors of negative control wells that contain PCR reagents but no target template and no IPC Notes Plus Minus Getting Started Guide for the 7300 7500 System 23 Creating a Plus Minus Plate Document Creating a New Plus Minus Plate Document You can enter sample information into a new plate document import sample information from existing plate documents or use a template document to set up new plate documents This section describes setting up new plate documents Refer to the Online Help for information about importing sample information or using template documents To create a new plus minus plate document 1 Select Start gt Programs gt Applied Biosystems 7300 7500 gt Applied Biosystems 7300 7500 SDS Software to start the 7300 7500 SDS software Select File gt New In the New Document Wizard click the assay drop down list then select Plus Minus assay Accept the default settings for the Container and Template fields 96 Well Clear and Blank Document Chapter 4 Performing the Plus Minus Pre Read Run Hew Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Plus Minus ba Container 96 well Clear Template Blank Document Browse Operator amp dministrator Comments 4 Inthe Plate Name field type Plus Minus Pre
59. riment The example experiment represents a typical plus minus experiment that you can use as a quick start procedure to familiarize yourself with the plus minus workflow Details about the plus minus workflow are described in the subsequent chapters of this guide Example Experiment boxes appear in subsequent chapters to illustrate workflow details The objective of the example plus minus experiment is to determine if an coli target sequence is present or not present in each batch of hamburger meat The experiment uses duplex PCR where a set of primers and a VIC labeled probe for the IPC plus a set of primers and a FAM labeled probe for the target E coli sequence are run together in each reaction The set of primers probe for detecting E coli was custom designed by Applied Biosystems Primer Express software Reactions were set up for PCR using the TaqMan Universal PCR Master Mix and appropriate primers and probes The example plus minus experiment data and results were generated using a 7300 7500 system by performing A pre read run on a plus minus plate to determine the baseline fluorescence associated with primers and probes before amplification An amplification run using an AQ plate document to generate real time PCR data which can be used to analyze and troubleshoot the PCR data for the plus minus assay if needed A post read run using the original plus minus plate document which automatically subtracts the pre read bas
60. s 14 0 90 to 250 nM and gloves probe and deionized water Total 45 0 4 Prepare the reaction plate a Pipette 45 uL of the reaction mixture into NTC plus IPC each well of a 96 well reaction plate b Pipette 5 uL of IPC block TE or water or unknown sample into the designated wells of a 96 well plate such as the example indicated in the table to the right see Samples Setting Up the Reaction Plate on Unknown pius Ww page 19 Note The final reaction volume in each well is 50 uL GR2363 Notes Plus Minus Getting Started Guide for the 7300 7500 System T Chapter 1 Introduction Example Plus Minus Experiment c Keep the reaction plate on ice until you are A Wells If preparing Add ready to load it into the 7300 7500 system dibus A1 to A6 NAC 5 uL of 10X Exo IPC Block A7 to A12 NTC 5 uL of 1X TE or H O B1 to H12 Us 5 uL of sample being tested for E coli a No Amplification Control Well contains no target template and no IPC b No Template Control Well contains no target template only IPC c Unknown Well contains both target template and IPC Perform the pre read run 1 Create a plus minus plate document Follow the instructions as described in Chapter 4 Briefly in the New Document ESTER Wizard Select n assay container and template Far the document and enter the
61. sk label unknown is used for both IPC and the target samples Notes 32 Plus Minus Getting Started Guide for the 7300 7500 System To create a new AQ plate document 1 Notes Select Start gt Programs gt Applied Biosystems 7300 7500 gt Applied Biosystems 7300 7500 SDS Software to start the 7300 7500 SDS instrument software Select File gt New The New Document dialog box opens In the Assay drop down list select Absolute Quantification Standard Curve Accept the default settings for the Container and Template 96 Well Clear and Blank Document Note A standard curve is not necessary for a non quantification amplification run In the Plate Name field type Plus Minus Amplification Click Next gt A list of detectors is displayed in the New Document Wizard Select the detectors to add to the plate document then click Add gt gt IMPORTANT Ensure that the reporter dye for the target detector is different from the reporter dye for the IPC detector which is VIC Note The TagMan Exogenous Internal Positive Control Reagents kit uses an IPC VIC labeled probe with TAMRA quencher Note To remove a detector in the Detectors in Document window select the detector then click Remove Creating a Plate Document for Sample Amplification Hew Document Wizard Define Document Select the assay container and template for the document and enter the operator
62. t Display to select the value to display Spectra Tab Displays the fluorescence spectra of selected wells The Cycles slider allows you to see the spectra for each cycle by dragging it with the pointer e The Cycle text box shows the current position of the slider Double clicking the y axis opens the Graph Settings dialog box where you can reset the y and x axes or allow autoscaling Notes B uy RB Slo EJ E ES Sr Yost tesa y Plate Y Spectra Y Component Amplification Plot Y Standard Curve Y Dissociation Y Report Setup Instrument y Plate Y Spectra Y Report Y ee E ng t t Pee rn 2891 RTL REX A Notemplate No template No template No template No template No template No template No template No template No template No tem NI NI NI NI NI NI N N NI NI INI INI uH uH uH uH uH Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknow mno nno mo o o o o o o mo mno m Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknow mno no o o o u e o no Oe no mo Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknow o no o o o o o o no o Do Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknow o o o o o o o o no Oe o
63. t read run the SDS software compares the relationship between the spectral changes in the unknown samples and the control reactions defined previously NAC and NTC An NAC threshold is calculated from the NAC control reactions and an IPC threshold is calculated from the NTC control reactions The IPC threshold is used to determine amplification of the unknown sample target signal for each well The NAC threshold is used to determine amplification of the IPC signal in each unknown sample well Calling Unknowns To call the unknown samples using the IPC the SDS software compares the normalized Using IPC reporter signals of each unknown sample to the IPC threshold and NAC threshold The results are determined as follows Ifthe unknown sample target signal is above the IPC threshold then the result is positive Ifthe unknown sample target signal is below the IPC threshold the SDS software compares the unknown target signal to the NAC threshold as follows fitis at or below the NAC threshold then the result is negative fit is above the NAC threshold then the result is called undetermined Notes Plus Minus Getting Started Guide for the 7300 7500 System 43 Chapter 6 Performing the Plus Minus Post Read Run Viewing Plus Minus Results The Plate Tab When the post read is complete select the f Setup Y instrument YResutts Plate Y Spectra Y Report Plate tab on the Results page to display t
64. tarting an amplification plate run 37 plate document amplification 32 creating 22 creating for AQ 33 parameters 22 sample names 26 Plate tab 42 44 53 plus minus assay about 4 60 amplification data 52 amplification run 10 calling unknowns using IPC 43 calls 22 chemistry kits 14 controls and unknowns 8 22 end point assay 32 final reaction volume 20 IPC 2 negative result 45 plate document 8 22 23 post read run 11 probe and primers 15 reaction mix 7 reaction plate 7 result calls 11 44 sample experiment 6 tasks 23 terms used 3 using absolute quantification for real time data 33 viewing results 43 workflow 4 plus minus plate documents importing sample information 27 templates 27 Post Read button 11 41 post read run analysis 11 performing 40 41 PrepMan Ultra Sample Preparation Reagent Kit 7 18 Pre Read button 9 29 pre readrun 8 29 creating plate document 24 performing 28 Primer Express software 7 15 probe and primers designing 15 Q quantification analysis terms 52 Quencher Dye drop down list 50 H reaction plate 7 real time PCR 32 reference passive 35 52 Report tab 55 reporter dye 52 Reporter Dye drop down list 50 required materials 5 result calls 56 Results tab 11 37 53 Rn vs Cycle view 54 Plus Minus Getting Started Guide for the 7300 7500 System Rn See normalized reporter ROX 35 o sample Experiment 15 18 19 20 27 42 44 45 46 50 sam
65. te into the instrument Note The A1 position is in the top left corner of the instrument tray 5 Click Start As the instrument performs the PCR run it displays real time status information in the Instrument tab and records the fluorescence resulting from cleavage of TaqMan probes in the presence of the target sequences After the run 1s finished the status values and the buttons are grayed out the Analysis button 1s enabled y and a message indicates whether or not the run is successful All data generated during the run are saved to the plate document that you specified in step 3 and can be analyzed later for troubleshooting purposes 6 To view real time PCR after the run is finished click the Analysis button select the Results tab select the Amplification Plot tab then select all wells in the upper left box next to A1 Note You can change the sample setup information sample name detector task after a run is complete Notes Performing the Amplification Run Save As Save in SDS Documents ck Ee My Recent Documents G Desktop My Documents My Computer My Network Places Cancel File name Save as type SDS Documents sds Y Keyed corner Plus Minus Getting Started Guide for the 7300 7500 System 37 Chapter 5 Generating Amplification Data Performing the Amplification Run Notes
66. tions text vii Ct vs Well Position view 55 Ct See threshold cycle Plus Minus Getting Started Guide for the 7300 7500 System Index D deltaRn 52 Delta Rn vs Cycle view 54 Detector Manager window 49 detector tasks 23 detectors 10 22 amplification run 33 creating 49 definition 49 removing 33 selecting 25 Detectors in Document window 25 DNA amplifying 10 52 chemistry systems 18 final concentration 18 isolation 7 18 quality 18 documentation feedback vili documents importing 27 plus minus plate document 22 templates 27 dyes 52 E exporting plate data 47 F FAM labeled probe 7 15 fluorescence spectra 22 53 fluorogenic 5 nuclease chemistry 14 probe 14 G Graph Settings dialog box 53 55 o9 Instrument tab 9 10 11 28 36 40 IPC amplification plot 45 blocked 25 34 blocking reagent 7 definition 2 TaqMan Exogenous Internal Positive Control Reagents Kit 2 VIC probe 2 VIC reporter dye 25 M manual Ct 51 MSDSs obtaining viii N New Detector window 49 New Document Wizard window 8 24 no amplification control 25 34 plot 44 See also NAC 3 20 22 no template control 26 34 See also NTC 3 20 22 normalized reporter 52 Notes field 50 nucleotide sequence 3 O OK amp Reanalyze button 51 omitting unused wells 27 online help 8 P passive reference 35 52 passive reference ROX 27 35 PCR cycles 14 duplex 6 reaction mix 20 real time 32 37 s

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