TaqMan® Exogenous Internal Positive Control
Contents
1. 7 095 935 8888 7 095 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 31 0 180 331409 To Reach Technical Support Through the Internet Telephone Fax Region Dial Dial United Kingdom 44 0 1925 825650 44 0 1925 282502 Warrington Cheshire All other countries not 44 0 1925 282481 44 0 1925 282509 listed Warrington UK Japan Japan Hacchobori 81 3 5566 6230 81 3 5566 6507 Chuo ku Tokyo Latin America Del A Obregon Mexico 305 670 4350 305 670 4349 We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products You can also order technical documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms then select the relevant support region for the produ2. Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493 17 18 Telephone Fax Region Dial Dial Eastern Asia China Oceania Australia Scoresby 61 3 9730 8600 61 3 9730 8799 Victoria China Beijing 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 2358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 750 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 420 2 61 222 164 420 2 61 222 168 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Poland Lithuania Latvia 48 22 866 40 10 48 22 866 40 20 and Estonia Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva
3. into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com AR Applied NB Biosystems Applera Corporation 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 03 2001 Part Number 4308335B an Applera business
4. make accurate assignments for any specific target Plate read detection is performed using the following instruments ABI PRISM 7700 Sequence Detection System ABI PRISM 7200 Sequence Detection System The Sequence Detection Systems from Applied Biosystems are used to measure the increase of reporter fluorescence following PCR Reporter signals are normalized to the emission of a passive reference Emission Intensity of Target Template Sequence Rn TT Emission Intensity of Passive Reference Emission Intensity of Internal Positive Control Rn pc a Emission Intensity of Passive Reference Materials and Equipment Kit Contents Core Kits Supplied by the User The TaqMan Exogenous Internal Positive Control Reagents P N 4308323 provide sufficient material to perform two hundred 50 L reactions There is enough 10X Block for twenty four 50 L reactions The kit contents are listed in the table below Component Volume Description 10X Exo IPC Mix 1 0 mL One tube containing IPC primers and probe 10X Exo IPC Block 1204L One tube containing enough blocking reagent for twenty four 50 L reactions 50X Exo IPC DNA 2004L One tube of IPC template DNA IMPORTANT The TaqMan VIC dye must be configured as a Pure Dye on the ABI PRISM 7700 7200 Sequence Detection Systems for it to appear on the Reporter pull down menu See User Bulletin 4 Generating New Spectra Components P N 4
5. 306234 pages 6 7 to configure TaqMan VIC as a Pure Dye One of the TaqMan core reagent kits listed in the following table is required in addition to the reagents supplied in the TaqMan Exogenous Internal Positive Control Reagents The Exogenous IPC Reagents have been optimized with the TaqMan Universal PCR Master Mix Application TaqMan Core Reagents Source PCR TaqMan Universal PCR Applied Biosystems Master Mix P N 4304437 TaqMan PCR Core Applied Biosystems Reagents P N N808 0228 reagents supplied Materials Supplied The items listed in the following tables are required in addition to the Equipment Item Source ABI PRISM 7700 Sequence Detection System ABI PRISM 7200 Sequence Detection System GeneAmp PCR System 9600 See your local Applied Biosystems representative for the instrument best suited to meet your needs Product Source Custom TaqMan Probes 5 000 pmol 15 000 25 000 pmol 50 000 100 000 pmol Applied Biosystems P N 450025 P N 450024 P N 450003 MicroAmp Optical 96 Well Reaction Plate and Optical Caps Applied Biosystems P N 403012 MicroAmp Optical 96 Well Reaction Plate Applied Biosystems P N N801 0560 MicroAmp Optical Tubes Applied Biosystems P N N801 0933 MicroAmp Optical Caps Applied Biosystems P N N801 0935 Deionized water or TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 Major laborato
6. TaqMan Exogenous Internal Positive Control Reagents VIC Probe Protocol AR 8SSm Copyright 2001 Applied Biosystems For Research Use Only Not for use in diagnostic procedures All rights reserved Printed in the U S A NOTICE TO PURCHASER DISCLAIMER OF LICENSE This product is optimized for use in the Polymerase Chain Reaction PCR and 5 nuclease detection methods covered by patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd No license under these patents to use the PCR process or 5 nuclease detection methods is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the PCR process for certain research and development activities accompanies the purchase of certain Applied Biosystems reagents when used in conjunction with an authorized thermal cycler or is available from Applied Biosystems 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 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 ABI PRISM Applied Biosystems and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AmpErase GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems Inc All other trademarks ar
7. ata Step Action 7 Examine the R values for the NTC wells in the FAM layer to confirm their reproducibility Note These wells are used to calculate the target threshold value 8 Examine the R values for the IPC wells in the VIC layer to confirm their reproducibility Note These wells are used to calculate the IPC threshold value 9 Click the Window button 10 Click the Event Log button 11 Examine the Event Log to follow the process by which the SDS 1 6 3 software identifies outliers and generates threshold values 12 Print the Experimental Report Note The FAM data from the NAC wells are not used in any calculations and usually these NAC wells are assigned No Amp This is designated by a in the analysis plate view In some instances however they may be assigned as target positive because of the addition of the IPC blocking solution to these wells This does not represent a problem and will not impact the correct assignment of unknown sample wells The ABI PRISM 7700 or 7200 Sequence Detectors determine positive or negative calls as described below Refer to your instruments user s manual for more information If the Detectable Target Template And the Detectable Then the Target FAM call is IPC VIC call is Template is a x No Amp a In the presence of a strong FAM signal for the target assay a negativ
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9. e assignment and or signal can be obtained in the VIC layer This results from the limiting primer concentrations used in the IPC assay Technical Support Contacting Technical Support To Contact You can contact Applied Biosystems for technical support by telephone or fax by e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below Contact technical support by e mail for help in the following product Technical Support areas by E Mail Hours for Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com Biochromatography PerSeptive DNA PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers tsupport appliedbiosystems com LC MS Applied Biosystems MDS Sciex apisupport sciex com or api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com In the United States and Canada technical support is available at the Telephone foll
10. e the sole property of their respective owners Contents Inttoductiol eese CES ERIS RESQUE E E WOCHEN ORE 1 Purpose of the Kit sacs cise teenie I iad Paige eh day idu 1 Simultaneously Amplifying Two DNAs lees 1 Interpreting Negative Results 0 0 0 00 02 e eee eee eee eee 2 Custom Applications eese susen uneer errereen rererere 2 End Point Detection and Post PCR Plate Read 3 Sequence Detection sc srein den e ed ba up eid eme td 3 Materials and Equipment 0 0 0 0 eee eee ee 4 Kit Contents eel Send tee ae eee DR Re Da RR n 4 Core Kits Supplied by the User 9 4 Materials Supplied by User 9 5 Storage and Stability l l 6 Preparing Reactions and Thermal Cycling 7 Introduction 52 cer e Ear eeu ws eh er ee 7 About Preparing Reactions 0 0 eee eee eee 7 Thermal Cycling stu RR Ree de nde Re Ge 8 Performing End Point Detection on the ABI PRISM 7200 or 7700 9 OVerVeWz od vs a Wr a i pO E n E PU HE De ERR PS 9 Setting up the Software 9 Setting Up the Plates dro re dE Ep eee 10 VEL VIC Wiz ess dta EP Ser ih gs C ERA sae eed MODI VES HT eS 10 FAM ayer 2 nies supe Ree deeds etes we 10 NICE AV GD s le ok Gash RR EUER Ie e Re A D eae 11 Analyzing Data for End Point Runs 0 00 00 eee eee eee 13 Analyzing Data s ai ia e a fed pe Rene 13 Target Template Calls 0 0 eee eee eee eee 14 Technical Support i i acces eet eee mee we
11. en RR US ean 15 Contacting Technical Support 9 15 To Contact Technical Support by E Mail 15 Hours for Telephone Technical Support 15 To Contact Technical Support by Telephone or Fax 16 To Reach Technical Support Through the Internet 19 To Obtain Documents on Demand llle eese 20 Appendix A Preventing Contamination 200 5 21 Introduction i creer pee eee Haha C weed ees am 21 AmpEr se UNG esp nekUDUU nt hos ERE CE PERDE A 21 General PCR Practices 2 0 0 ee eee e 22 Appendix B References 0 0 0 cece cee cece ees 23 Introduction Purpose of the Kit Simultaneously Amplifying Two DNAs The TaqMan Exogenous Internal Positive Control Reagents is a pre optimized internal positive control IPC which can be spiked into samples to distinguish true target negatives from PCR inhibition The kit is designed to Distinguish types of negative results A negative call for the target sequence and positive call for the IPC indicates that no target sequence is present A negative call for the target sequence and negative call for the IPC suggests PCR inhibition Avoid amplification of endogenous genes Permit coamplifcation of the IPC and the target sequence without compromising amplification of the target sequence Perform optimally with the TaqMan Universal PCR Master Mix IMPORTANT To obtain a
12. es with PCR Nature 339 237 238 Lakowicz J R 1983 Energy Transfer In Principles of Fluorescent Spectroscopy Plenum Press N Y pp 303 339 Lawyer F C Stoffel S Saiki R K Myambo K B Drummond R and Gelfand D H 1989 Isolation characterization and expression in Escherichia coli of the DNA polymerase gene from the extreme thermophile Thermus aquaticus J Biol Chem 264 6427 6437 Lee L G Connell C R and Bloch W 1993 Allelic discrimination by nick translation PCR with fluorogenic probes Nucleic Acids Res 21 3761 3766 Livak K J Flood S J A Marmaro J Giusti W and Deetz K 1995 Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization PCR Meth Appl 4 357 362 Longo N Berninger N S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Lyamichev V Brow M A D and Dahlberg J E 1993 Structure specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases Science 260 778 783 23 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches
13. n Sequencing Systems 1 800 831 6844 then press 32 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Product or Product Area Telephone Dial Fax Dial Voyager MALDI TOF Biospectrometry and Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 13 1 508 383 7855 Biochromatography BioCAD Workstations and Poros Perfusion Chromatography Products 1 800 899 5858 then press 14 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 15 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 15 1 508 383 7855 PNA Custom and Synthesis 1 800 899 5858 then press 15 1 508 383 7855 FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 16 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 Applied Biosystems MDS Sciex 1 800 952 4716 1 650 638 6223 Outside North America Telephone Fax Region Dial Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 South
14. n the File New Plate Select single reporter plate read and the correct instrument 7700 or 7200 Define the FAM layer as shown in FAM Layer on page 10 See your instrument user s manual for more information Define the VIC layer as shown in VIC Layer on page 11 See your instrument user s manual for more information Click the Show Analysis button Click the Post PCR Read button The software will perform the Plate Read Note The TaqMan Exogenous Internal Positive Control Reagents are designed to utilize the post PCR plate read function Utilization of the pre PCR plate read may interfere with the ability of the system to make accurate assignments for any specific target Save the plate Proceed to Analyzing Data for End Point Runs on page 13 Setting Up the Plates 10 Overview The plate setup for the FAM layer and the VIC layer are shown FAM Layer The FAM layer consists of the following see figure below Six No Amplification Control NAC wells Six No Target Template Control NTC wells Eighty four Unknown UNKN wells IMPORTANT Six replicates of No Template Control must be run to make calls at a 99 7 confidence level These are required to accurately define the thresholds for the FAM and VIC layers o El Sample Type UNKN Unknown 7200 Single Reporter Sample Name Comment Replicate Sho
15. or positive FAM and VIC calls on the basis of the No Template Control NTC and the Negative Internal Positive Control IPC baselines In this kit the FAM layer shows the positive and negative calls for the target template and the VIC layer shows the calls for the IPC The target template calls are made on the following basis If the Detectable Target Template And the Detectable Then the Target FAM call is IPC VIC call is Template is a No Amp a In the presence of a strong FAM signal for the target assay a negative assignment and or signal can be obtained in the VIC layer This is a result of the limiting primer concentrations used in the IPC assay The IPC DNA primers and probes supplied with this kit can be used with all sample target systems Refer to the TaqMan Universal PCR Master Mix Protocol P N 4304449 for instructions on how to optimize your target system s performance End Point Detection and Post PCR Plate Read Sequence Detection The TaqMan Exogenous Internal Positive Control Reagents are designed for Plate Read end point detection only Plate Read detection collects one fluorescent scan per tube after PCR is completed The TaqMan Exogenous Internal Positive Control Reagents are designed to utilize the post PCR plate read function Utilization of the pre PCR plate read may interfere with the ability of the system to
16. owing times Technical Support Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time 15 To Contact In North America Technical Support To contact Applied Biosystems Technical Support use the telephone or by Telephone or fax numbers given below To open a service call for other support Fax needs or in case of an emergency dial 1 800 831 6844 and press 1 16 Product or Product Area Telephone Dial Fax Dial ABI PRISM 3700 DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 21 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 22 1 650 638 5981 Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 23 1 650 638 5981 Integrated Thermal Cyclers ABI PRISM 877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 ABI PRISM 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 Biolnformatics includes BioLIMS BioMerge and SQL GT applications 1 800 831 6844 then press 25 1 505 982 7690 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 31 1 650 638 5981 Protein Sequencing Procise Protei
17. quested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery Appendix A Preventing Contamination Introduction AmpErase UNG Due to the high throughput and repetitive nature of the 5 nuclease assay special laboratory practices are necessary in order to avoid false positive amplifications Kwok and Higuchi 1989 This is because of the capability for single DNA molecule amplification provided by the PCR process Saiki et a 1985 Mullis et a 1987 AmpErase UNG uracil N glycosylase UNG is a pure nuclease free 26 kDa recombinant enzyme encoded by the Escherichia coli uracil N glycosylase gene This gene has been inserted into an E coli host to direct expression of the native form of the enzyme Kwok and Higuchi 1989 UNG acts on single and double stranded dU containing DNA It acts by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The enzyme causes the release of uracil thereb
18. r Total 45 JA 4 5 mL Pipet 5 7A of sample into each well of a 96 well plate as shown in FAM Layer on page 10 Note The final reaction volume in each well should be 50 AL Well IF preparing Then add A1 A6 NAC 5 4A of 10X Exo IPC Block A7 A12 NTC 5 pL of 1X TE B1 H12 UNKN 5 LA of sample Thermal Cycling Use the following procedure to amplify samples Step Action 1 Place the MicroAmp Optical 96 Well Reaction Plate in the thermal cycler 2 Program the thermal cycler Thermal Cycler Times and Temperatures Initial Steps Each of 40 Cycles Melt Anneal Extend GeneAmp PCR HOLD HOLD CYCLE System 9600 or 2 min 10 min 15 sec 1 min 9700F 50 C 95 C 95 C 60 C ABI PRISM 7700 HOLD HOLD CYCLE Sequence 2 min 10 min 15 sec 1 min Detector 50 C 95 C 95 C 60 C a If the 9700 thermal cycler is used use the 9600 emulation mode 3 Perform PCR amplification Store the PCR products at 2 6 C until you are ready for analysis Performing End Point Detection on the ABI PRISM 7200 or 7700 Overview To perform end point analysis on the ABI PRISM 7200 or 7700 Sequence Detectors follow the procedure described below Setting up the To set up the Sequence Detection System software Software Step Action 1 Open the ABI PRISM Sequence Detection System SDS software 2 Double click o
19. ry suppliers MLS Note The ABI PRISM 7700 and ABI PRISM 7200 Sequence Detectors use the MicroAmp Optical 96 Well Reaction Plate and MicroAmp Optical Caps IMPORTANT Sequence Detector Do not use MicroAmp Optical Tubes with the ABI PRISM 7200 Storage and Store the TaqMan Exogenous Internal Positive Control Reagents at 20 Stability to 25 C However if the reagents will be consumed within one month store them at 2 4 C If stored under the recommended conditions the product will maintain performance for one year from time of receipt Preparing Reactions and Thermal Cycling Introduction The TaqMan Exogenous IPC Reagents are designed for use with end point detection only IMPORTANT To obtain assignments with a 99 7 confidence level a post PCR plate read should always be performed Real time document assignments on the ABI PRISM 7700 Sequence Detector have not been verified to be statistically significant About Preparing Prepare the reactions as described below Follow precautions to Reactions prevent PCR contamination as described in Appendix A on page 21 Step Action 1 Make the following Master Mix and pipet 45 Z L into each well of the 96 Well Reaction Plate Volume for Volume for 100 Item one Reaction Reactions TaqMan Universal PCR Master 25 JA 2 5 mL Mix 10X Exo IPC Mix 5 UL 0 5 mL 50X Exo IPC DNA 14L 0 1 mL Target primers probe and 14 4L 1 4 mL deionized wate
20. ssignments with a 99 7 confidence level a post PCR plate read should always be performed Real time document assignments on the ABI PRISM 7700 Sequence Detector have not been verified to be statistically significant By using the TaqMan Exogenous Internal Positive Control Reagents a low copy target DNA can be amplified in the same tube with the IPC Although the target and IPC DNAs may differ in initial copy number the amplification efficiency of the target reaction is not compromised This is achieved by limiting the concentration of IPC primers in the PCR reaction In the PCR reaction the IPC is detected using a VIC labeled probe and the target template is detected using a FAM labeled probe Interpreting Negative Results Custom Applications The TaqMan Exogenous Internal Positive Control Reagents in conjunction with your target system identify samples that are positive and negative for a specific target sequence The kit distinguishes between two types of negative reactions Samples identified as negative because they lack the target sequence Samples identified as negative because of the presence of a PCR inhibitor During amplification the sample and IPC generate reporter fluorescence signals such that identification calls may be made on unknown samples Positive and negative calls are made on the basis of statistical analysis of data from the two dye layers The statistical analysis sets up threshold values f
21. ternal Negative IPC Reporter pop up menu Click OK The SDS software returns to the Setup Plate view 11 Vic Layer Sample Type Setup The following assignments should then be made in the Sample Type Setup to the VIC layer see figure below Six Internal Positive Control Negative IPC wells corresponding to the FAM layer NAC wells Ninety Internal Positive Control Positive IPC wells corresponding to the FAM layer NTC and UNKN wells Analyzing Data for End Point Runs Analyzing Data To analyze data Step Action 1 Click the Show Analysis button on the setup window 2 Click the Instrument Advanced Options button The Advanced Options dialog box appears SS Advanced Options Yiewer L Display mse in Multicomponent View x Display best fit in Raw Spectra View Analysis Spectra Components gog Use background in Spectra Components folder gog Use pure spectra in Spectra Components folder Miscellaneous Options g Set 7700 Exposure Time g Use Spectral Compensation for Real Time x Use Spectral Compensation for Endpoint Ifusing the ABI PRISM 7700 select the Use Spectral Compensation for End Point option If using the ABI PRISM 7200 do not select the Use Spectral Compensation for End Point option Click OK Click Analyze Click Display R from the Analysis menu 13 14 Target Template Calls To analyze d
22. w Analysis Dye Layer 1 4 VIC Layer The default layer for IPC assignments in the SDS v 1 6 3 software is the JOE dye layer These assigments must be changed to the VIC dye layer before using the Taqman Exogenous Internal Positive Control Reagents To set up the IPC assignments for use with a VIC probe Step Action 1 From the Dye Layer pop up menu select VIC 2 From the Sample Type pop up menu select Sample Type Setup IPC Internal Positive Control IPC Internal Positive Control Sample Type Setup Sample Type Not In Use The SDS software displays the Sample Type Setup dialog box From the Internal Positive IPC Reporter pop up menu select VIC Acronym Name Color Reporter ipc Internal Positive IFc Internal Negative sto Standard ui Unknown rc No Template Control Nac No Amplification nec No Probe Control The SDS software displays VIC in the Reporter box for the Internal Positive Control entry as shown below Acronym Hame Color Feporter Internal Positive IMPORTANT The TaqMan VIC dye must be configured as a Pure Dye on the ABI PRISM 7700 7200 Sequence Detection Systems for it to appear on the Reporter pull down menu See User Bulletin 4 Generating New Spectra Components P N 4306234 pages 6 7 to configure TaqMan VIC as a Pure Dye Repeat Step 3 to select VIC from the In
23. y creating an alkali sensitive apyrimidic site in the DNA The enzyme has no activity on RNA or dT containing DNA 21 22 General PCR Use the following precautions to minimize sample cross contamination Practices and PCR product carryover Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Use positive displacement or air displacement pipettors with filter plugged tips Change tips after each use Keep reactions and components capped as much as possible Clean lab benches and equipment periodically with 10 bleach solution Appendix B References Forster V Th 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Ann Phys Leipzig 2 55 75 Holland P M Abramson R D Watson R and Gelfand D H 1991 Detection of specific polymerase chain reaction product by utilizing the 5 to 3 exonuclease activity of Thermus aquaticus DNA polymerase PNAS USA 88 7276 7280 Kwok S and Higuchi R 1989 Avoiding false positiv
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