User Manual revision A6
Contents
1. Run Recovery Power cycling Recover last experiement Accessing Log Files Identifying Serial and Version Numbers Appendix A Concepts and Glossary Concepts Glossary Technical Assistance Declaration of conformity The copyright of this instruction book is the property of Bibby Scientific This instruction book is supplied by Bibby Scientific on the express understanding that it is to be used solely for the purpose for which it is supplied It may not be copied used or disclosed to others in whole or part for any purpose except as authorised in writing by Bibby Scientific Bibby Scientific reserves the right to alter change or modify this document without prior notification Bibby Scientific Limited Beacon Road Stone Staffordshire ST15 OSA United Kingdom Tel 44 0 1785 812121 Fax 44 0 1785 810405 e mail enquiries techne com www techne com PrimePro rev A 6 Page 28 29 29 30 30 30 30 Si 31 31 32 32 33 36 37 Polymerase Chain Reaction PCR denotes the amplification of DNA templates catalyzed by DNA polymerase in the presence of primers dNTPs divalent cations like Mg 2 and a buffer solution The ability to visualize and quantify the amplification of DNA as it occurs during PCR Is called Real Time PCR or Quantitative PCR qPCR This is made possible by the use of fluorescent chemistries an optical system that can capture the emitted fluorescence at every PCR cycle and softwa2. Assay 1 Channel 2 Assay 2 Channel 3 Assay 3 Channel 4 Assay 4 Log of starting quantity target Figure 7 Standard Curves for Four Multiplexed Assays The Prime Pro 48 includes two excitation LED arrays 452 486 nm and 542 582 nm and four filter channels Table 1 which enable detection of up to four separate targets in a single reaction Figure 7 Prime Pro 48 is factory calibrated for certain dyes within each channel marked in Table 1 but also supports additional dyes that are excited and detected within the instrument specifications IA PrimePro rev A 6 2 1 UNPACK THE PRIME PRO 48 1 Remove the computer and accessories from the box 2 Lift the Prime Pro 48 instrument out of the crate Place it on a flat surface and remove the foam packaging NOTE Keep the box and packaging in case of a return 3 Check to ensure that all components are present and intact Your system comes with Accompanying literature 2 2 PLACE PRIME PRO 48 ON THE BENCH Prime Pro 48 requires 5 cm 2 inches of unimpeded space at the front and back for ventilation and 7 5 cm 3 inches above the instrument so that the lid can be opened safely Make sure you have easy access to the power switch on the lower right back corner of the Prime Pro 48 instrument and that there are two wall outlets 100 240 VAC 50 60 Hz 5A within 2 m 6 feet of the instrument Figure 8 Prime Pro 48 Space requirements 2 3
3. For Genotyping experiments Select a reporter dye for Alleles 1 and 2 Select a Quencher Quencher molecules absorb fluorescent emissions of reporter dyes when in close proximity By default the quencher is set to None for DNA binding dye chemistry and Non fluorescent for Hydrolysis probes NOTE BHO and MGB are considered non fluorescent quenchers NOTE Fluorescent quenchers such as Tamra are not recommended for use in the Prime Pro 48 For Genotyping experiments Select a quencher for Alleles 1 and 2 e For Relative Quantification experiments Specify the PCR Efficiency 4 Click go close the Assays dialog box and return to Plate Layout Airay Mama PCR Efficiency 9 Actay Atay Figure 15 Assay Dialog Box Relative Quantification Experiments 5 For Relative Quantification experiments Select at least one Reference assay 6 For all experiment types If you want to use controls select a control type for each assay from the Role drop down list Options for roles in the drop down list change according to the type of experiment NOTE The Role you assign has no affect on the analysis calculations of your experiment The Role is just a label for your convenience For example if you want to define an assay as a no reverse transcription control or you want to define an assay as a control sample that you know to be negative for the target you are amplifying you can select the Role Negative or NTC Data from the
4. Prime Pro 48 system is set up and ready to use register your Prime Pro by going to www techne com warranty asp and completing a short questionnaire Registering your Prime Pro ensures that you will receive software updates in the future While you are visiting the web site take advantage of the following online resources to support your research Prime Pro Customer Support knowledge database warranty information webinars and seminar series www techne com Online Ordering www techne com Tradeshows workshops and meeting presence www techne com or contact your local dealer ow PrimePro rev A 6 3 1 PRIME PRO 48 WORKFLOW 1 Prepare the sample plate load it into the Prime Pro 48 and close the lid See Load the Plate on page 12 Z Launch the Prime Pro 48 software on the PC 3 Define and name the experiment by selecting the application detection chemistry starting material and specific method for your application See Define a New Experiment on page 13 TIP To use a pre defined thermal profile and plate layout for your experiment click Templates and select one of the template experiments saved on your computer 4 Review the thermal profile and adapt it if needed See Set Up the Thermal Profile on page 14 5 Set up the plate layout by defining assays samples and standards and assigning them to wells See Define the Plate Layout on page 15 6 Start the run The Monitor Run tab opens See Monitor Run on page 21 WARNING Do not ope
5. negative control or NTC is not used in calculations to normalize the data Z For Genotyping and High Resolution Melt experiments If you want controls with unique names that are not included with the software set them up from the Options menu After controls are created they are available for use in the Role drop down list in the Assays section a Click the Options menu b Click Control Types c Use the arrows to select the Number of Control Types d Select the Color To change the color select the round color icon next to the assay name Double click a new color in the color palette e An Abbreviation automatically populates f Click OK 8 Proceed to set up samples PrimePro rev A 6 3 10 SET UP SAMPLES Samples Nurnber of Samples 3 yr Sample Name Standard Unknown NTC El Apply Sample Color Figure 17 Sample Dialog Box On the Plate Layout tab click ps or Set Up Samples to open the Samples dialog box Use the arrow keys to select the number of samples For each sample define a name and color Click CA o close the Samples dialog box and return to Plate Layout For Relative Quantification and HRM experiments Select at least one Reference sample Proceed to assign assays and samples to wells O 01 A W N S ot PrimePro rev A 6 3 11 ASSIGN ASSAYS AND SAMPLES TO WELLS Figure 18 Plate Layout Tab Assigning Assays and Samples 1 Left click and drag the mouse to highlight one or more wells o
6. 36 5 2 RUN RECOVERY If the Prime Pro 48 instrument loses connection with the computer the Prime Pro 48 often continues to run After you reconnect your computer with the Prime Pro 48 you can usually retrieve the run data because the data file of your last experiment is stored in the instrument To Recover the run data follow the Power Cycling directions below to turn the computer and the Prime Pro 48 off and on After Power Cycling select Options Recover Last Experiment in the main menu 5 3 POWER CYCLING 1 Turn off the power on the Prime Pro 48 The power switch is on the back of the instrument 2 Shut down the computer so that the power is off 3 Wait three minutes NOTE Make sure that the Prime Pro 48 and the computer are powered off for three minutes before you proceed to step 4 4 Turn on the Prime Pro 48 instrument 5 Turn on the computer 6 Double click the Prime Pro 48 icon on the computer desktop to start the Prime Pro 48 software Communication between the computer and the Prime Pro 48 instrument is established within five minutes NOTE If you are power cycling Prime Pro 48 because the Prime Pro 48 instrument and the computer lost connection a warning message might appear during the connection time that says the experiment file may not have completed The message asks you if you want the software to attempt to Recover the run Click Yes 7 When Status Instrument Connected displays at the bottom of the screen the co
7. AM 542 582 604 644 KORC 452 486 562 5960 HEX VIC 542 582 665 705 Cy5 0670 a If you use ROX as a passive reference for normalization your plate layout cannot include an assay whose reporter dye is measured in channel 2 PrimePro rev A 6 ER 3 8 ASSAYS AND REPORTER DYES FOR GENOTYPING When defining a plate layout genotyping experiments need special setup of assays and reporter dyes Assign at least one well for each homozygous Allele 1 Wild Type homozygous Allele 2 Mutant and tor heterozygous controls Make sure to select a different reporter dye for each Allele See Genotyping and High Resolution Melt on page HER Figure 15 Example Plate Layout for a Genotyping Experiment 3 9 SET UP ASSAYS 1 On the Plate Layout tab click Q or Set Up Assays to open the Assays dialog box 2 Use the arrow keys to select the number of assays 3 For each assay Define a name and color For Genotyping experiments Click the yellow box C which appears after setting role as Wild Type Heterozygous or Mutant this will let you further define the control type name and will automatically define an abbreviation meme en smn e tex texa Select a Reporter dye thereby setting the channel If your dye is not listed select a reporter with the most similar excitation and emission range to your dye refer to the Channel table on Multiplexing Real Time PCR on page 8 ot PrimePro rev A 6
8. CONNECT PRIME PRO 48 1 Connect one end of the Ethernet cable to the Ethernet port on the computer Connect the other end to the Ethernet port on the rear panel of the Eco 48 A Equipment must be connected to reliable and suitable protective earth connection 2 Connect the Eco 48 power cord to the AC power inlet on the rear panel and then to the wall outlet B A suitably approved mains power cord set may be used It must be ensured that the cord set meets the host country requirements 3 Connect the computer power cord to the wall outlet Figure 9 Prime Pro 48 Connection C PrimePro rev A 6 s 2 4 TURN ON THE PRIME PRO 48 1 Turn on the computer wait up to five minutes for Microsoft Windows to boot fully then turn on the Prime Pro 48 instrument A The instrument runs a series of self tests that take up to 20 minutes 2 At any time after turning on the instrument double click the Prime Pro 48 icon on the computer desktop to start the Prime Pro 48 software B Communication between the computer and the Prime Pro 48 instrument is established within five minutes When the READY indicator lights on the front panel stop flashing and remain solid the instrument is ready 3 Open the Prime Pro 48 by pressing the round silver button on the front to raise its handle while lifting the handle from the bottom until the Prime Pro 48 pops open C Figure 10 Prime Pro 48 Startup Sequence 2 5 REGISTER YOUR PRIME PRO 48 Once your
9. EE READY STATUS 2O OD ERROR Sees READY CT STATUS OOOOO ERROR Er z PrimePro rev A 6 Description Power on Initializing Conducting self tests and heating the thermal block Ready Idle Run In Progress Do not switch off or open the lid while a run is in progress Fatal Error Run Terminated Error light flashing Instrument may have overheated or encountered a hardware failure Camera Initialization Error 2 long flashes of the Error light Try power cycling the instrument to resolve Temperature Sensor Error 4 long flashes of the Error light Hardware problem Call customer support Lights READY STATUS 60000 ERROR READY SS STATUS OOO 010 ERROR SSS READY Ce STATUS 22 OS TS eS ERROR CE READY ooo o o STATUS 90000 ERROR E READY CT STATUS OOOOO ERROR ETT Description Run Complete Communication to PC error Try power cycling the instrument to resolve Software Updating Non Fatal Error Decide whether you want to terminate the run Hardware Failure Solid Error light Hardware problem Call customer support Camera Trigger Error 3 long flashes of the Error light Try power cycling the instrument to resolve Temperature Response Error 5 long flashes of the Error light Hardware problem Call customer support 4 2 MINIMUM SYSTEM REQUIREMENTS Operating The following
10. Operation Manual PrimePro rev A 6 1 TT LLL 11111 ALTT AT TT LLL TTT bobby INTENDED USE The Prime Pro 48 Real Time PCR System is intended to support the Real Time polymerase chain reaction PCR application needs of life science researchers This includes gene expression quantification and analysis as well as genotyping by allelic discrimination or high resolution melting The system is able to support other applications and protocols as well Prime Pro 48 features high quality optical and thermal modules to provide optimal performance and data quality The system includes data analysis software that is preloaded on a computer and provided on a separate USB drive for installation on additional computers as needed Additional accessories and consumables are provided or available tor purchase to ensure the best user experience Use of the Prime Pro for specific intended uses such as polymerase chain reaction PCR Real Time qPCR or high resolution melting HRM may require the user to obtain rights trom third parties It is solely the user s responsibility to obtain all rights necessary for the intended use of Prime Pro 48 This document and its contents are proprietary to Prime and its affiliates and are intended solely for the contractual use of Its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any ot
11. afety requirements for electrical equipment for measurement control and laboratory use General requirements EN6101 2 010 2003 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 010 Particular requirements for laboratory equipment for the heating of materials CE mark affixed 2014 Other Standards CFR47 Part 15 2013 Radio Frequency devices Subpart B Class A UL 61010 1 2012 Electrical Equipment For Measurement Control and Laboratory Use Part 1 General Requirements UL 61010A 2 010 2002 Electrical Equipment for Laboratory Use Part 2 Particular Requirements for Laboratory Equipment for the Heating of Materials CAN CSA C22 2 No 61010 1 2012 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General requirements CAN CSA C22 2 No 61010 2 010 2004 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials o gt z de Mr 5 Marriott Signed sli iv Technical Director Bibby Scientific Ltd Bibby Scientific Ltd Stone Staffs ST15 OSA UK Bibby Scientific Tel 44 0 1785 812121 Fax 44 0 1785 813748 PrimePro rev A 6 Bibby Scientific Bibby Scientific Ltd Beacon Road Stone Staffordshire ST15 OSA United Kingdom Tel 44 0 1785 812121 Fax 44 0 1785 813748 e mail info bi
12. allelic discrimination assays using hydrolysis probes provide a rapid and sensitive method to geno type samples These assays can refer to a single nucleotide polymorphism SNP or insertion deletion assays Two variants alleles are interrogated at the same time multiplex qPCR Most frequently one probe is labeled with a FAM dye and the other with a VIC dye Samples with FAM signal and no VIC signal are homozygous for allele 1 samples with VIC signal and no FAM signal are homozygous for allele 2 and samples with both FAM and VIC Signal are heterozygous Figure 5 Figure 5 Allelic Distribution Scatter Plot Figure 6 HRM Difference Plot High Resolution Melt HRM enables the detection of almost any genetic variation SNPs mutations Because HRM assays only require primers and a dye no probes or DNA sequencing the method is simpler and cheaper than traditional genotyping approaches After the amplification phase the amplicon is slowly heated until it melts The melting temperature and profile are directly linked to the amplicon sequence HRM s power comes from the resolution of the sample s melt profile It requires a high quality optical system and precise thermal uniformity HRM PCR amplicons below 300 bp provide the best resolution The shape of the resulting melting curves which is sensitive to almost any genetic change determines sample identity To easily cluster equivalent samples a reference curve e g Wild Type is subtracted fro
13. are pending US patents 13 417121 12 753806 14 620998 European patents 7783905 8 12178744 4 10759513 4 10007637 1 and India patent 9450DELNPO8 Further patents are pending PrimePro rev A 6 Revision History Table of Contents Chapter 1 Overview Real Time PCR Absolute and Relative Quantification Genotyping and High Resolution Melt Multiplexing Real Time PCR Chapter 2 Setup Unpack the Prime Pro 48 Place Prime Pro 48 on the Bench Connect Prime Pro 48 Turn on the Prime Pro 48 Register your Prime Pro 48 Chapter 3 Workflow Prime Pro 48 Workflow Load the Plate Define a New Experiment New session tab Set Up the Thermal Profile Define the Plate Layout Assays and reporter dyes Assays and reporter dyes for genotyping Set up assays Set up samples Assign assays and samples to wells Define standards Auto calculate serial dilutions Manually enter dilutions Assign dilutions automatically Assign dilutions manually Monitor Run Export Results and Data Templates and Sample Sheets Making a Sample Sheet for Import Chapter 4 System Information Components System Requirements Specifications and Environmental Requirements Page 3 CO N DD UW UWI O O LO MO WO WO zi 11 12 is 14 14 15 15 16 16 18 19 20 20 20 20 21 21 22 23 24 25 25 27 i PrimePro rev A 6 ER Symbols Electromagnetic Compatibility Cleaning And Maintenance Chapter 5 Troubleshooting Introduction
14. are the minimum requirements for running the Pro 48 software e Operating system must be Windows 7 not starter and in UK or USA English Either 32 or 64 bit variant are compatible e Memory RAM 4GB DDR3 e Communication Gigabit Ethernet and USB 2 0 e Processor Core 13 3rd generation with at least 1 7 Ghz clock speed ideally 2 4GHz clock speed Example PC Dell Vostro 2420 or direct equivalent The system should have all firewalls disabled as a precaution Some firewalls disrupt communication between the PC and the Prime Pro 48 The PC running the Prime Pro hardware should be a designated unit with no 3rd party software on the system Network ports being used by the Prime Pro 48 should be set to not allow the operating system to shut down the port to save energy Instructions for PC set up can be found on the Prime Pro 48 quick Start guide that shipped with the unit 4 3 SPECIFICATIONS AND ENVIRONMENTAL REQUIREMENTS Optical Thermal Operational Physical Environmental Light Source Detector Thermal Cycling Thermal Uniformity Sample Format Reaction Volume Warmup Time Typical PCR Run Time Sensitivity of Detection High Resolution Melt Multiplexing Passive Reference Dimensions Weight Electrical Temperature Range Humidity Range Protection Two sets of 48 LEDs 452 486 nm and 542 582 nm CCD camera 4 filters 505 545 nm 562 596 nm 604 644 nm and 665 705 nm Proprietary hollow silver
15. ation of PCR Xn is described by the following equation Xn Xo 1 E n where X number of target molecules at cycle n X initial number of target molecules E efficiency of target amplification and n number of cycles Amplification efficiency E is described by the following equation E 10 re 1 The acceptable range of assay efficiency 90 to 110 or a slope between 3 1 and 3 6 A slope of 3 32 indicates 100 efficiency meaning that the number of template molecules doubled in each PCR cycle Common reference genes o High expression 18S ribosomal RNA 18S Beta actin ACTB Beta 2 microglobulin B2M glyceraldehyde 3 phosphate dehydrogenase GAPDH and phosphoglycerokinase PGK Medium expression Transferrin receptor TfR o Low expression Transcription factor IID TATA binding protein TBP and glucuronidase GUS Always validate your reference genes to ensure that the genes you select are stable in your experiments El PrimePro rev A 6 6 2 GLOSSARY Term Absolute Quantification Allelic Discrimination Amplicon Assay Baseline used to Channel Ci Cq Dark Quencher Delta Rn ARn Derivate Melt Curve DNA Binding Dye dsDNA Dual Labeled Hydrolysis Probe Dynamic Range Efficiency Endogenous Control End Point Analysis Exogenous Control FAM 6 carboxy fluorescein Filter Definition An assay that quantifies unknown samples by interpolating their qua
16. ature control and thermal uniformity across the sample plate Standard Fast protocol performs 40 PCR cycles in approximately 40 minutes Motors Stirring Paddles Silver hollow Thermal Block containing the conductive fluid 4 1 2 OPTICAL SYSTEM Two LED arrays provide individual sample well excitation Four detection filters support almost all PCR chemistries and multiplex detection ROX is optional CCD camera acquires high quality data in all wells and filters at each PCR cycle Factory calibrated optics support SYBR Green FAM HEX VIC ROX Cy5 and Q670 dyes You can also use other dyes that are compatible with the excitation and emission wavelengths U O U gt CCD Camera Filter Slide Green LED Array Blue LED Array Figure 28 Prime Pro 48 Thermal System Mi gt ib SE ni P mi E Figure 29 Prime Pro 48 Thermal System PrimePro rev A 6 ER 4 System Information 4 1 3 LIGHTS The Pro 48 has three indicator lights on the front Ready Status and Error The following tables show the meaning of each combination of off on and flashing lights If Power Cycling is advised for an error light see Power Cycling on page 30 Lights READY C o STATUS OOOO ERROR SVT EEE Zz STATUS OOOO O ERROR CD READY eas STATUS OJOJOO ERROR CD READY a STATUS QT HU Z S OT NN CD READY CT STATUS CHOC CC ERROR C
17. ays and samples to wells See Assign Assays and Samples to Wells on page 19 Define standards Standard Curve Quantification experiments only See Define Standards on page 20 Select the Rox Normalization checkbox if you are using Rox passive reference dye to normalize across your plate Um B WN You can lay out the plate any time between defining the experiment and analyzing the data However you will only be able to see deconvoluted data while monitoring the run 3 7 ASSAYS AND REPORTER DYES An assay is the set of primers or primers probe used to quantify a nucleic acid target sequence Assays can have different roles such as Unknown Standard Negative Positive or NTC Non Template Controls Each assay is associated with a reporter dye which identifies the assay during analysis Reporter dyes can belong to one of four channels each of which is defined by a specific excitation and emission range You can assign up to four assays per well Within each well assays cannot use reporter dyes from the same channel see following table If they did data trom assays using the same channel would be indistinguishable during analysis A red outline around a well indicates that it contains more than one reporter dye from the same channel and requires correction before you can analyze your data Table 2 Channels and Reporter Dyes Excitation nm Emission nm Fluorophores Calibrated on the Pro 48 Reporter 452 486 505 545 SYBR Green F
18. bby scientific com www bibby scientific com Bibby Scientific Middle East Ltd BPO Box 27842 Engomi 2433 Nicosia Cyprus Tel 357 22 660 423 Fax 357 22 660 424 e mail sales bibby scientificme com www bibby scientific com Bibby Scientific France Batiment le Deltaparc Silic Paris Nord 2 7 rue du Canal BP 55437 Villepinte 95944 ROISSY Charles de Gaulle France Tel 33 0 1 48 63 78 00 Fax 33 0 1 48 63 78 01 Email ventes bibby scientific fr www bibby scientific com Bibby Scientific US Ltd Bibby Scientific Singapore 3 Terri Lane Suite 10 Prudential Tower Level 26 Burlington NJ 08016 30 Cecil Street Singapore 049712 USA Tel 65 6631 2976 Tel 800 225 9243 Fax 44 0 1785 810405 Fax 609 589 2571 Email info bibby scientific com www bibby scientific com www bibby scientific com
19. block with Peltier based system Q0 1 C 48 well plate 5 20 ul 20 minutes Less than 40 minutes for 40 cycles 1 copy Supported resolution to 0 1 C Detection of up to four targets simultaneously four plex Optional ROX 34 5 cm Wx 31cmDx32cmH 13 6 in W x 12 2 in D x 12 6 in H 13 6 kg 30 Ib including power supply 100 240 VAC 50 60 Hz 5A Operating 15 C to 40 C 59 F to 104 F Storage 10 C to 100 C 50 F to 212 F Operating 15 90 Relative Humidity Storage 5 95 Relative Humidity IP20 PrimePro rev A 6 4 System Information 4 4 SYMBOLS The Prime Pro 48 has three indicator lights on the front Ready Status and Error The following tables show the meaning of each combination of off on and flashing lights If Power Cycling is advised for an error light see Power Cycling on page 30 Symbol Description Symbol Description Do Not Throw in Trash At end of useful life recycle the system or device EC REP European Representative Fuse replacement fuses must meet the stated rating CAUTION Hot Surface Humidity Range on packaging Manufactured By indicates acceptable shipping and storage limits Mark of European Conformity Model Number device complies with the EMC Directive 2004 108 EC and the Low Voltage Directive 2006 95 EC i UH Serial Number Temperature Range on packaging indicates acceptable shipping and storage limits a Prim
20. but unassigned to a layout re ProStudy Before and after a but not during a run See directions for Making a Sample Sheet for Import Import Plate Template A plate template is a plate layout and does not include a thermal profile de ProStudy Before and after a run but not during a run The Plate tab must be active Export Plate Template A plate template plate is a plate layout thermal and does not include a thermal plate profile CSV ProStudy Before and after a run run but not during a run PrimePro rev A 6 ER 3 18 MAKING A SAMPLE SHEET FOR IMPORT To create your own sample sheet for import into Prime Pro 48 use a program like Excel to create a csv file The sample sheet m Calibri can contain up to 99 assay names and 48 sample names F Format Painter B Z 1 In the same column enter the heading Assay Name k Clipboard and Sample Name 2 Make sure there is a space between the assay list and 4 the sample list Assay Name 3 Give each assay and sample a unigue name Assay 1 4 Save the file as a csv file Assay 2 Assay 3 Make sure there is a space Sample Name between the Sample 1 assay list and Sample 2 sample list Sample 3 Figure 27 Example of a Sample Sheet a PrimePro rev A 6 4 1 1 COMPONENTS THERMAL SYSTEM w gt Proprietary hollow silver thermal block filled with circulating conductive fluid provides Superior temper
21. ds used to quantity nucleic acids by qPCR are the absolute and relative quantification methods The absolute quantification method is based on a standard curve generated from serial dilution of a nucleic acid template of known concentration Figure 3 Quantification of unknown samples is determined by interpolat ing the sample Ca from the standard curve Throughout the rest of this document absolute quantification is referred to as a standard curve experiment Slope Efficiency R Log of starting quantity target Figure 3 Five Point 10 Fold Standard Curve The slope of the standard curve measures the efficiency of the assay E 10 Ysepel 1 A slope outside the ac ceptable range slope 3 1 to 3 6 and E value between 90 and 110 typically indicates a problem with the template or assay design The R2 value a measure of reaction performance should be gt 0 99 for the assay to accurately quantify unknown samples The relative quantification method measures the level of gene expression in a sample relative to the level of expression of the same gene in a reference sample In addition the level of expression of every gene in the assay is normalized to the expression of a reference gene The results RQ value obtained are expressed as relative levels or fold change in gene expression compared to the reference or control sample Figure 4 Figure 4 Relative Quantification Experiment KA PrimePro rev A 6 Genotyping
22. e format you chose to output If a type of data is not available it appears grayed out in the Export Options area In the Export Options dialog box select the desired file format and components to export then click 2 PrimePro rev A 6 P H efiefiefiefielief ie elielielieli ded dd dl dddddd ld ef ef ef fed ef ef ed s Adelei A Alee Figure 25 Plate Layout View in the Monitor Tab Export Options mmm G Exceriners Fie Name Evaluation Plate Exot Location C Program Piles amp 6 PCRimax Eco Browse a CV _ Sv Qo PDF Report Excel Export Options O Component Data E Plate Layout O Component Meli Dala E Thermal Prolia n on Figure 26 Export Options dialog box 3 Workflow 3 17 TEMPLATES AND SAMPLE SHEETS The table below explains the differences between a template a plate template and a sample sheet It also describes where and when templates and sample sheets can be saved imported and exported The commands below are available under the File menu in Prime Pro 48 Only Import Plate Template and Export Plate Template are available in Prime Pro 48 Study Save a Template Command Description A template is a layout and profile ecot ProStudy File Extension Where When Before a run After saving the template is available for use It Is listed in the Templates tab on startup in Pro 48 Import a Sample Sheet A sample sheet is a list of assays and samples
23. ePro rev A 6 4 5 ELECTROMAGNETIC COMPATIBILITY This equipment complies with the emission and immunity requirements described in IEC 61326 1 2005 and IEC 61326 2 6 2005 To confirm proper operation The electromagnetic environment should be evaluated prior to operation of the system Do not use this system in close proximity to sources of strong electromagnetic radiation e g unshielded intentional RF sources as these may interfere with proper operation If you notice any interference discontinue using the system until all issues are resolved Resolution may include moving cords from other equipment away from the system plugging the system into an outlet on a different circuit from other equipment or moving the system away from the other equipment If you continue to have difficulties contact Prime 4 6 CLEANING AND MAINTENANCE Clean the block and housing as needed following these directions CAUTION If hazardous or biohazardous material is spilled onto or into the equipment clean it immediately 1 Turn the system off and allow the block to cool completely 2 Using a lint free cloth slightly dampened with clean water gently wipe the surfaces of the equipment If a stronger cleaning agent is needed use a lint free cloth slightly dampened with 95 isopropyl alcohol Follow these practices for proper maintenance of your Prime Pro 48 Every time you use the system visually check it to confirm there is no obvious phy
24. elative Quantification Reporter Dye Rn Normalized Reporter Signal ROX carboxy X rhodamine Slope Definition dsDNA binding agents typically attached to the 3 end of hydrolysis probes MGBS increase the Tm value of probes thus leading to smaller probes Hairpin probes containing a sequence specific loop region flanked by two inverted repeats A quencher dye at one end of the molecule quenches the reported dye at the other end Sequence specific binding leads to hairpin unraveling and fluorescent signal generation Simultaneous analysis of more than one template in the same reaction An assay with all necessary components except the template The use of control genes with a constant expression level to normalize the expression of other genes in templates of variable concentration and quality A fluorescence dye such as ROX that the software uses as an internal reference to normalize the reporter signal during data analysis Element used for heating and cooling in a qPCR machine Molecule that absorbs fluorescence emission of a reporter dye when in close proximity BHQ is a quencher The coefficient of correlation between measured Cq values and the DNA concentrations It is a measure of how closely the plotted data points fit the standard curve The closer to 1 the value the better the fit R2 is ideally gt 0 99 A passive dye or active signal used to normalize experimental results Genes with a wide and constant level of expre
25. face to ensure the seal is secure 8 Place the plate adapter with your loaded and sealed plate into a compatible centrifuge rotor along with the seconds supplied plate adapter for balance Centrifuge the plate at 250 g for 30 seconds Do not spin more than 500 g Verify that there are no air bubbles at the bottom of the wells 9 Open the Prime Pro 48 lid and place the plate on the block aligning the notch against the top left corner WARNING Forcing the plate into any other orientation could damage the instrument WARNING Be careful not to touch the heated lid above the plate It heats to 105 C 221 F when the instrument is turned on and could result in burns 10 Close the Prime Pro 48 lid The heated lid automatically creates a seal around and on top of the plate to prevent evaporation 11 Proceed to Define a New Experiment Ni PrimePro rev A 6 3 3 DEFINE A NEW EXPERIMENT 1 Double click the Prime Pro 48 icon on the desktop to open the software The New Experiment tab opens Figure 11 Main Real Time PCR Chemistries 2 Click an Application Option that is the specific method or protocol you want to use for your experiment When you select the application the software automatically configures options for downstream setup and analysis For example High Resolution Melt HRM is associated with DNA Binding dyes and so the other three detection chemistries are grayed out for High Resolution Melt experiments Experime
26. ght a Standard Assay well and click enoti the Assign button beside the appropriate a dilution quantity Figure 22 Unit Dalina Staniarda Assign Quantity ig i pe ml h E L ROR NOT m ae ML 3 18 MONITOR RUN WARNING Do not open the lid while a run is in progress This allows extraneous light into the system and will corrupt the data NOTE If you have not yet defined the plate layout Define the Plate Layout on page 15 you will only be able to view progress against the Thermal Profile on this tab Select assays to view in Amplification Plot B Shows current stage of the Thermal Profile highlighted in orange Figure 23 Monitor Tab C Amplification View shows deconvoluted data in real time for each well D Amplification Plot shows deconvoluted data in real time for selected wells Amplification Plot Highlight wells in the Amplification View to display a subset of data in the main graph Figure 24 Amplification Plot PrimePro rev A 6 A Plate layout view shows sample type sample identity dilution and assays B Select by assay sample or call using the drop down arrow Select the identity of the assay using the drop down arrow D Turn Show Plate Layout on and off by selecting the box OO 3 18 EXPORT RESULTS AND DATA Results as well as other data can be exported by selecting File Export in the main menu The options available here vary depending on the type of experiment you ran and the fil
27. her purpose and or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Prime Prime does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT S INJURY TO PERSONS INCLUDING TO USERS OR OTHERS AND DAMAGE TO OTHER PROPERTY Prime DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT S DESCRIBED HEREIN INCLUDING PARTS THEREOF OR SOFTWARE OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY Prime IN CONNECTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH USE ONLY PrimePro rev A 6 2010 2014 Bibby Scientific All rights reserved This instrument is covered by US patents US8003370 US8008046 US8232091 US9068947 US8987685 US8035811 Germany patents DE202010010523 4 DE2120100000393 China patents ZL201020267691 9 ZL201090000931 7 and US registered design USD653764 The following patents
28. increase in the electronic state of a photon containing molecule Carboxy 2 4 4 5 7 7 hexachlorofluorescein An enhancement of the traditional melt curve analysis which increases the detail and information captured A probe that is not hydrolyzed by Tag polymerase Hybridization probes can be used for melt curve analysis Examples include Roche FRET and Molecular Beacons A probe that is hydrolyzed by the 5 endonuclease activity of Tag polymerase An exogenous control added to a multiplex gPCR assay to monitor the presence of inhibitors in the template Carboxy 4 5 dichloro 2 7 dimethoxyfluorescein Light Emitting Diode A light that is illuminated by the movement of electrons in a semiconductor material LED lights do not have filaments that burn out and do not get very hot A view of an amplification plot using linear dRn values y axis versus PCR cycles x axis A view of an amplification plot using log dRn values y axis versus PCR cycles x axis A self quenched fluorogenic primer and a corresponding unlabeled primer When the primer is incorporated into DNA during PCR the fluorophore is de quenched leading to an increase in fluorescent signal See Derivative Melt Curve 6 2 GLOSSARY Term Minor Groove Binders MGBs Molecular Beacons Multiplexing No Template Control NTC Normalization Passive Reference Peltier Quencher R2 Coefficient of Correlation Reference Reference Genes R
29. m the other curves to generate a difference plot Figure 6 Captions added to illustrate the different genotypes only Resources Livak KJ 1999 Allelic discrimination using fluorogenic probes and the 5 nuclease assay Genet Anal Biomol Eng 14 143 149 POLAND server http Awww biophys uni duesseldort de local POLAND poland html Wojdacz TK Dobrovic A Hansen LL 2008 Methylation sensitive high resolution melting Nature Protocols 3 12 1903 1908 PrimePro rev A 6 The simultaneous detection of multiple targets in a single reaction is called multiplexing An advantage of multiplexing is that it conserves sample allowing more data to be obtained from the same amount of material Another advantage is that multiplexing permits the inclusion of an internal control reference assay for normalization purposes significantly increasing data precision Channel Dye Channel 1 A 505 545 nm Channel 2 A 604 644 nm SYBR Green FAM ROX Texas Red HEX JOE TET VIC Gy 5250676 Channel 3 562 596 nm Channel 4 665 705 nm Table 1 Examples of Prime Pro 48 Compatible Dyes Validating a multiplex qPCR assay can be challenging The advent of more advanced qPCR master mixes has significantly reduced the amount of optimization typically required making multiplex qPCR a much more attractive alternative Validation of assays using a standard curve Is a must to ensure data accuracy Channel 1
30. modify it based on your reagent s recommended protocol You can set up cycle parameters in the Thermal Profile at any time after defining the experiment but only before starting the run Click Q to add a new stage such as a reverse transcription incubation at the beginning or additional PCR Cycling stages The stage will appear at the end of the cycle and you can drag it to the desired location Alternatively you can drag the Q icon to the location within the profile where you would like the new stage to be added The camera icon 9 indicates when the Pro 48 collects image data In multi step PCR you can select whether to collect data at the annealing or extension step Extension is the default To move it to annealing mouse over the annealing step and click the dim camera icon that appears Only one step can be designated to collect image data To remove a stage drag it to the 4 trash can or highlight it and press Delete a PrimePro rev A 6 3 6 DEFINE THE PLATE LAYOUT Annealing Annealing intercalating dyes Hydrolysis probes SYBR Green TaqMan Figure 14 Plate Layout Tab The Plate Layout tab lets you define how your samples assays and standards are laid out on the plate loaded in the Prime Pro 48 The analysis software uses the plate layout to calculate data values Plate layout involves the following steps 1 Set up assays See Set Up Assays on page 16 Set up samples See Set Up Samples on page 18 Assign ass
31. mputer and the instrument are connected 8 Wait until the Ready light on the Prime Pro 48 instrument is solid blue This takes up to twenty minutes PrimePro rev A 6 5 4 RECOVER LAST EXPERIMENT After you follow the directions for Power Cycling you can Recover the experiment that was running when the connection was lost between the Prime Pro 48 Instrument and the computer Select Options Recover Last Experiment in the main menu The name of your latest experiment with the word Recovered appears in the New Sessions tab at the bottom of the screen To confirm that your data is Recovered go to the Monitor Run tab If your data is Recovered you will see the data in the Amplification Plot and the Amplification View Select File Save As to save your Recovered data 5 5 ACCESSING LOG FILES Prime Pro 48 stores a log file that helps with many troubleshooting issues Prime Technical Support may ask for this file To access the log file follow the procedure here Pro LO CON O UT Turn on the Prime Pro 48 and the computer Double click the Prime Pro 48 icon on the computer desktop to start the Prime Pro 48 software Communication between the computer and the Prime Pro 48 instrument is established within five minutes When Status Instrument Connected displays at the bottom of the screen the computer and the instrument are connected Close the Prime Pro 48 software on the computer Navigate to C ProgramFiles Prime Prime P
32. n the lid while a run is in progress This allows extraneous light into the system and will corrupt the data 7 When the run is complete open the Prime Pro 48 lid Press the plate release lever and remove the plate from the block Dispose of any hazardous materials in biohazard caustic material or other appropriate containers according to your local safety regulations Cer ba forra p p Dey er ae g Ma nein jad ee ee a Ma maiia Men zar pou Current eco Ma rar f Da pira ip VA bas rol bam Mai PrimePro rev A 6 ER 3 2 LOAD THE PLATE 1 Thaw all necessary reagents templates primers probes and master mix 2 Turn on the PC then the Prime Pro 48 and wait until the Pro 48 Ready light is solid blue o Confirm that the block and optical path are clear of visible contaminants and there is no physical damage to the system such as dents frayed cords or damaged levers 4 Place a 48 well plate into the Prime Pro 48 sample loading dock aligning the notch with the matching indentation on the adapter 5 Turn on the dock light and incline the dock to a comfortable angle for pipetting 6 Pipette samples and gPCR reagents into the plate according to your protocol WARNING Wear protective gloves and eyewear when handling any material that might be considered caustic or hazardous 7 Seal the plate with an Prime Pro 48 optical seal Holding the plate in place on the Prime Pro 48 sample loading dock drag the squeegee firmly across the sur
33. n the plate layout diagram Wells turn yellow when they are highlighted as shown in columns 1 and 2 of Figure 18 2 Click the Assign button for up to four assays and one sample in the left pane of the window to assign the assays and sample to the highlighted wells 3 To change the role of an assay in a given well highlight the well and then select the desired Assay Role from the drop down list NOTE For quantification experiments that will be combined using the Prime Pro 48 Study software for at least one plate in the study you must specify e Standard Curve studies At least two wells with the role Standard but with different quantities e Relative Quantification studies At least one well with the role Unknown or Positive and a sample assigned Any plate meeting these specifications can be used as the mother plate in your study The mother plate Is the plate against which the other experiments in the study will be compared 4 For Standard Curve experiments Proceed to define standards For other experiments Click to start the run now ia nr Sample Name and Colour gt Assay Role and Colour O a de 20000 Quantity gt Multiplexed Well with 3 dyes gt To clear setting highlight and press Delete Figure 19 Well in the Plate Layout PrimePro rev A 6 E 3 12 DEFINE STANDARDS When you set an Assay Role to Standard a small orange Standards butt
34. nt Type Options Quantification Relative Quantification or Standard Curve Genotyping Genotyping PCR or Genotyping Single Read High Resolution Melt PCR with HRM Curve or HRM Curve Only Table 1 Examples of Prime Pro 48 Compatible Dyes 3 Select a Detection Chemistry Detection Chemistry Type NUPCR Probe based detection chemistry DNA Binding Dye SYBR green assays Hydrolysis Probe 5 nuclease assays Other Non hydrolitic assays 4 Select a Starting Material 5 Enter an experiment name of up to 20 characters 6 Click z The Setup window opens with the Thermal Profile tab visible PrimePro rev A 6 3 4 NEW SESSION TAB Use the New Session tab to create open and select multiple experiments The tab is on the bottom left of the screen To add a new experiment select the page with a star icon You can also right click on the New Session tab to rename an experiment make a new experiment open an experiment or close an experiment Figure 12 New Session Tab 3 5 SET UP THE THERMAL PROFILE A Drag to move the stage B Double click the temperature plateau to adjust temperature and duration C Type new temperature D Drag the bar up and down to adjust the temperature E Data collection point F Type new cycle time G Toggle two three four or five step PCR H Click or type to add or remove cycles When you define the experiment a corresponding default thermal profile is selected automatically You can use this or
35. ntities from a standard curve based on a serial dilution of a sample containing known concentration An assay that discriminates between two alleles gene variants A fragment of DNA synthesized by a pair of primers during PCR The set of primers or primers probe used to quantify an amplicon The initial PCR cycles when little fluorescence signal is generated This will be subtract the background The combination of excitation and emission spectra used to monitor amplification for a given assay Threshold Cycle See Ca Quantification Cycle The cycle number at which the fluorescent signal crosses the threshold It is inversely correlated to the logarithm of the initial copy number A quencher without any native fluorescence Black Hole Quencher BHQ dyes are an example The normalized Fluorescence of an amplification plot with background and ROX normalization dye correction A plot of temperature x axis versus the derivate of fluorescence with respect to temperature dF dT y axis Used to analyze the Tm of an amplicon A dye that increases its fluorescence in the presence of double stranded DNA Double stranded DNA See hydrolysis probe The range of template concentration over which accurate Cq values can be determined Extrapolation is not recommended See Slope An RNA or DNA template that is spiked into each sample at a known concentration Qualitative analysis of PCR data at the end of PCR Allelic discrimination assay
36. on appears to the right of the assay role 1 Click St open the Set Up Standards pane in the lower left of the window 2 Select the units that are used in your standards and then enter the quantity 3 13 AUTO CALCULATE SERIAL DILUTIONS 1 To auto calculate serial dilutions click Define Standards Figure 20 Well in the Plate Layout The Dilutions dialog box opens 2 Enter the number of points in the standard curve the Nabea Porte Statina Quantic Oddion Fasie quantity of the most concentrated standard and the fe 600 10 Z desired dilution factor and then click 3 14 MANUALLY ENTER DILUTIONS 1 Enter the value of the first standard into the first Quantity field below Units 2 Press Enter to commit the value and open the next Quantity field 3 15 Assign Standard Dilutions to Wells You can assign standard dilutions to wells manually or automatically 3 16 ASSIGN DILUTIONS AUTOMATICALLY 1 Left click and drag the mouse over a group of Standard Assay wells Vertical Wells Points on Standard Curve Horizontal Wells Replicates The Apply Standards button becomes active when you have selected a suitable group of wells 2 Click WApphuStandardaj The dilutions and replicates are automatically added in the highlighted group of wells eee mm er ong mn ser ong ne ze eee Figure 21 Selecting Standard Assay Wells ow PrimePro rev A 6 3 17 ASSIGN DILUTIONS MANUALLY Highli
37. re that can quantify the amplification The two most commonly used gPCR chemistries are DNA binding dyes and hydrolysis probes Figure 1 DNA binding dyes fluoresce when bound to double stranded DNA Hydrolysis probes fluoresce when the reporter molecule is removed trom its quencher molecule by the 5 exonuclease activity of DNA polymerase Annealing Annealing intercalating dyes Hydrolysis probes SYBR Green TagMan Figure 1 Main Real Time PCR Chemistries Little fluorescence is generated during initial PCR cycles Figure 2 Data from these early cycles define the baseline for the assay Initial As fluorescence approaches the level of optical detection the reaction reaches the exponential phase which is the region where the Cq is determined Ca is the PCR cycle at which the fluorescent signal crosses the detection threshold level and is used for quantification Finally as reaction components are consumed and amplicons become abundant the generation of additional fluorescent signal slows down and eventually reaches a reaction plateau 4 Plateau 2 Exponential Fluorescence Threshold Line ee eee eee eee 1 Initial Figure 2 The Three Phases of gPCR Resources Saiki RK Scharf S Faloona F Mullis KB Horn GT Erlich HA Arnheim N 1985 Science 230 1350 1354 Higuchi R Fockler G Dollinger G and Watson R 1993 Biotechnology N Y 11 1026 1030 PrimePro rev A 6 The two primary metho
38. ro and find the GetLogs exe file If you did not install the Prime program on the C drive your drive letter may be different Open the GetLogs exe Tile Select the Include Previously Uploaded Logs check box Click Get Log Files When the file finishes transferring Done appears on the GetLogs dialog box Save the zipped log file on your computer in an easy to remember place Email the zipped log file to Prime Technical Support at technehelpObibby scientific com 5 6 IDENTIFYING SERIAL AND VERSION NUMBERS To identity the Prime Pro 48 instrument ID serial number the software version number and the instrument firmware version number select Help About Prime Pro 48 License Information on the main menu PrimePro rev A 6 ER 6 1 CONCEPTS The weight of one genome g size of genome in bp x 618 g mol bp x Avogadro s number One human genome g 3 x 10 bp x 618 g mol bp x 6 02 x 1023 3 08 x 101 g mol One haploid cell sperm egg 3 08 pg of DNA One diploid cell 6 16 pg of DNA There is approximately one copy of every non repeated sequence per 3 08 pg of human DNA The average cell contains 10 20 pg of total RNA About 90 95 of total RNA is rRNA 18S 5 8S and 28S 1 3 is mRNA RNA concentration ug ul A DNA concentration ug ul A 40 D 1000 where D dilution factor and A absorbance at 260 nm x60 20 D 1000 where D dilution factor and A absorbance at 260 nm The exponential amplific
39. s genotyping are an example An RNA or DNA template that is spiked into each sample at a known concentration The most commonly used reporter dye at the 5 end of a hydrolysis probe Components used to limit the bandwidth or the excitation or emission energy to the next component of the optical path PrimePro rev A 6 ER 6 2 GLOSSARY Term Fluorophore Fluorescence HEX High Resolution Melt HRM Hybridization Probe Fluorophore Fluorescence HEX High Resolution Melt HRM Hybridization Probe Hydrolysis Probe Internal Positive Control IPC JOE LED Linear View Log view LUX Primer Set Melt Curve PrimePro rev A 6 Definition The functional group of a molecule that absorbs energy at a specific wavelength and emits it back at a different wavelength The immediate release of energy a photon of light as a result of an increase in the electronic state of a photon containing molecule Carboxy 2 4 4 5 7 7 hexachlorofluorescein An enhancement of the traditional melt curve analysis which increases the detail and information captured A probe that is not hydrolyzed by Tag polymerase Hybridization probes can be used for melt curve analysis Examples include Roche FRET and Molecular Beacons The functional group of a molecule that absorbs energy at a specific wavelength and emits it back at a different wavelength The immediate release of energy a photon of light as a result of an
40. sical damage such as dents frayed cords or damaged levers If you see any damage discontinue use and contact Techne Technical Support technehelp bibby scientific com Once a year run a known test sample to confirm accurate analysis CAUTION The Prime Pro 48 contains materials that may be hazardous to the environment if not disposed of properly Be sure to dispose of materials according to all local state provincial and national regulations PrimePro rev A 6 ER 5 1 INTRODUCTION For most errors on the Prime Pro 48 an on screen message opens with instructions for correcting the error Error lights on the front of the instrument may also indicate a problem and how to fix it See Lights on page 47 If connection is lost between the Prime Pro 48 instrument and the computer use the Run Recovery section to Recover your run data See Run Recovery on page 30 This Troubleshooting section also has directions on how to access files and information that will help Prime Technical Support troubleshoot your problem For instance if you contact a Prime Technical Support representative they might ask for copies of run specific files for troubleshooting purposes such as a log file For technical questions visit the Prime Pro 48 support pages on the Prime website for access to frequently asked questions For problems with run quality or performance contact Prime Technical Support For more information see Technical Assistance on page
41. ssion Typically used to normalize the expression of other genes Examples of commonly used reference genes 16S 18S GAPDH and b actin An assay used to measure the expression of a target gene in one sample relative to another sample and normalized to a reference gene Fluorescent dye used to monitor amplicon accumulation This can be a dsDNA binding dye or a dye attached to a probe Each dye is associated with a certain channel Reporter fluorescent signal divided by fluorescence of the passive reference dye The most commonly used passive reference dye The slope of a standard curve It is a measure of assay efficiency E 106159 1 where a slope of 3 32 is equal to 100 efficiency E or an exact doubling of template molecules in each PCR cycle Acceptable efficiencies range from 3 6 90 to 3 1 110 Overly high efficiencies indicate qPCR inhibition usually due to contaminants in the sample Overly low efficiencies typically indicate problems with the reaction mix concentration PrimePro rev A 6 ER 6 2 GLOSSARY Term Standard Standard Curve Standard Deviation SD TAMRA Target Template Threshold TET Tm Unknown Y Intercept 6 3 TECHNICAL ASSISTANCE Definition A serial dilution of a target of known concentration used as template to generate a standard curve A plot of Cq values against the log of target amount Used to determine an assay s dynamic range efficiency slope R2 and sensitivit
42. y y intercept The SD of replicate Cg measurements is a measure of the precision of the assay Tetramethyl 6 carboxyrhodamine Commonly used as a quencher The DNA or RNA sequence to be amplified See Target Template can also refer to a saved experiment that can be used as a model for new experiments in the software A level set above the background signal generated during the early cycles of qPCR When adjusted manually it should be set in the middle of the exponential stage of qPCR Carboxy 2 4 7 7 tetrachlorofluorescein The temperature at which 50 of dsDNA is single stranded melted A sample containing an unknown amount of template In a standard curve the value that crosses the y axis at x 1 single copy target For technical assistance go to www techne com or contact your local dealer MSDSs Material safety data sheets MSDSs are available on the Prime website at www techne com Product Documentation Product documentation you can obtain PDFs from the Prime website www techne com NA PrimePro rev A 6 Declaration of Conformity Real time Thermal Cycler Models PrimePro 48 This product complies with the requirements of the EU Directives listed below 2004 108 EC EMC Directive 2006 95 EC Low Voltage Directive L V D Compliance with the requirements of these Directives is claimed by meeting the following standards EN61326 1 2013 Emissions Class A and Immunity Basic Requirements EN61010 1 2010 S
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