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TaqMan® Universal Master Mix II Protocol

1. Item NUR Custom TagMan Small scale human 40X 1000 x 5 uL rxns 4331349 x EME Small scale non human 40X 1000 x 5 uL rxns 4332077 Medium scale human 40X 3000 x 5 uL rxns 4332072 Medium scale non human 40X 3000 x 5 uL rxns 4332075 Large scale human 80X 12 000 x 5 uL rxns 4332073 Large scale non human 80X 12 000 x 5 uL rxns 4332076 TaqMan Pre Small scale 40X 1500 x 5 uL rxns 4351379 elena Medium scale 40X 5000 x 5 pL rxns 4351376 Assays Large scale 80X 12 000 x 5 uL rxns 4351374 TaqMan Validated and Coding Genotyping Assays 4331183 Small scale 20X concentration 750 x 5 uL rxns TaqMan CYP2C19 2 400 rxns 4312561 dep as kel CYP2C9 2 400 rxns 4312559 for Allelic CYP2C9 3 400 rxns 4312560 Discrimination CYP2D6 3 400 rxns 4312554 CYP2D6 4 400 rxns 4312555 CYP2D6 6 400 rxns 4312556 CYP2D6 7 400 rxns 4312557 CYP2D6 8 400 rxns 4312558 TaqMan Drug Metabolism Genotyping Assays 8 Tris EDTA TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 made using AM9849 DNase free RNase free sterile filtered water DNAZap Solution 2 x 250 mL bottles AM9890 RT PCR Grade Water 10 x 1 75 mL bottles AM9935 DNase free water AM9914G t Includes CD with protocol Assay Information File AIF DME Assay Index and Troubleshooting Guide 8 Go to www appliedbiosystems com then search TaqMan Drug Metabolism Assay Plastics not
2. Primer 3 gt g 5 5 3 D Reverse Primer Figure8 Completion of polymerization 60 TagMan Universal Master Mix ll Protocol Appendix E Safety This appendix covers m General chemical Safety JW kk kk kK KK KK KK KK KK KK KK KK KK KK KK 62 Bib ur eii EN P222 63 B Chemical wastesafety sss kk sk a k klk cette eens 64 m Biological hazard safety WA kk kk kK KK KK KK KK KK KK KK KK KK KK KK 65 B Chemical alerts 0 0 0 KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK K k 65 TaqMan Universal Master Mix Il Protocol 61 Appendix E Safety General chemical safety General chemical safety Chemical hazard warning Chemical safety guidelines 62 WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions AN WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument A WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in
3. 14 TaqMan Universal Master Mix II Protocol Section 1 Gene expression quantitation Analyze the results Table 2 Correct and incorrect baseline settings Baseline set correctly The amplification curve begins after the maximum baseline Baseline set too low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value Baseline set too high The amplification curve begins before the maximum baseline Decrease the End Cycle value Analyze the data You can perform two types of quantitation using the TaqMan Universal Master Mix II Relative quantitation compares the relative expression of a target gene between an unknown and a reference sample You can perform relative quantitation using the standard curve method or the comparative Cy method Absolute quantitation compares the C of an unknown sample against a standard curve with known copy numbers Quantitation of cDNA Gene expression can be measured by comparing the relative expression of a target oe to a calibrator gene in a unknown sample and in a physiological reference sample In a typical sample experiment gene expression levels are studied as a function of either a treatment of cells in culture of patients or of tissue type The calibrator sample in each case is the cDNA from either the untreated cells or patients or a specific tissue type All quantitations are also normalized to an endoge
4. 2 Step Kit Applied TaqMan RNA to C 2 Step Kit Mini Pack PN 4399902 Biosystems e TagMan RNA to C 2 Step Kit 1 Pack PN 4399367 TaqMan MicroRNA Reverse Transcription Kit Applied e 1000 reactions PN 4366597 Biosystems e 200 reactions PN 4366596 50 TaqMan Universal Master Mix II Protocol Appendix B Ordering Information Optional user supplied reagents for gene expression quantitation Optional user supplied reagents for gene expression quantitation For a description of these reagents go to www ambion com techlib index Materials Source MagMAX AI ND Viral RNA Isolation Kit 50 purifications AM1929 MagMAX Viral RNA Isolation Kit 50 purifications AM1939 mirVana miRNA Isolation Kit 40 purifications AM1560 RecoverAll Total Nucleic Acid Isolation Kit for FFPE 40 purifications AM1975 RiboPure Bacterial Kit AM1925 RiboPure Blood Kit 40 purifications AM1928 RiboPure RNA Isolation Kit 50 purifications AM1924 RiboPure Yeast Kit AM1926 RNAlater ICE Solution 25 mL AM7030 RNA ater Solution 100 mL AM7020 RNAqueous 4PCR Kit 30 purifications AM1914 RNAqueous Kit 50 purifications AM1912 RNaseZap RNase Decontamination Solution 250 mL AM9780 RT PCR Grade Water 10 1 75 mL bottles AM9935 TRI Reagent 100 mL AM9738 Turbo DNA free 50 reactions AM1907 TaqMan Universal Master Mix Il Protocol 51 Appendix B Ordering Info
5. 7500 Fast or 7900HT Fast instruments use Standard mode thermal cycling conditions If you use assays other than the TaqMan Gene Expression assays or use thermal cycling conditions other than those specified in this protocol validate your assays and re optimize your thermal cycling conditions as needed Refer to Real Time PCR Systems Chemistry Guide PN 4348358 for more information on selecting thermal cycling conditions TaqMan Universal Master Mix Il Protocol 11 TaqMan Universal Master Mix II Perform real time PCR amplification Run the plate 12 Refer to the appropriate instrument user guide for detailed instructions on loading and running the PCR plates See Related documentation on page 69 for a list of user documentation for Applied Biosystems real time PCR systems To run the plate 1 In the system software open the plate document or experiment that corresponds to the reaction plate 2 Load the reaction plate into the real time PCR system 3 Start the run TaqMan Universal Master Mix II Protocol Section 1 Gene expression quantitation Analyze the results Analyze the results Baseline and threshold values Automatic calculation of the baseline and threshold The general process for analyzing gene expression data involves a Viewing the amplification plots for the entire plate b Setting the baseline and threshold values to determine the threshold cycles C for the amplificatio
6. Applied Protocol E Biosystems 2009 2010 Life Technologies Corporation All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT AL LOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Notice to Purchaser License Disclaimer This kit conveys no patent rights expressly or by implication under any patent or patent application owned by or licensable by Life Technologies Cor poration that covers any thermal cycling instrument apparatus or system any composition reagent or kit or any process Specifically but without limitation no right immunity authorization or license is granted expressly or by implication for the processes of reverse transcription PCR or the 5 nuclease assay NOTICE TO PUCHASER LIMITED LICENSE A license to perform the patented 5 Nucl
7. For example Select File Open Spot Set Right click the sample row then select View Filter View All Runs User attention words Two user attention words appear in 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 How to obtain support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can e vi Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs 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 TaqMan Universal Master Mix II Protocol Protocol TaqMan Universal Master Mix II Product information Purpose of the product TagMan Universal Master Mix II is a convenient mix of components except primers probes template and water necessary to perform a real time polym
8. 10 15 20 25 30 35 40 e Figure 1 Typical amplification curve TaqMan Universal Master Mix Il Protocol 13 TaqMan Universal Master Mix II Analyze the results Manual setting of the baseline and threshold If you use the system software to set the baseline and threshold values manually for any detector assay in the study perform an adjustment procedure for each detector assay Refer to your real time PCR system documentation for guidance on manually setting and adjusting your threshold and baseline Table 1 Correct and incorrect threshold settings Threshold set correctly The threshold is set in the exponential phase of the amplification curve Threshold settings above or below the optimum increase the standard deviation of the replicate groups ARn 0 01 0 001 1X 0 0001 Threshold set too low The threshold is set below the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Set the threshold up into the exponential phase of the curve An 0 1 0 01 gt 0 001 1X7 0 0001 Threshold set too high The threshold is set above the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Set the threshold down into the exponential phase of the curve 11 754743 001 0 0001
9. 2 mL 1000 tubes in strips of eight PN 4316567 MicroAmp Optical 8 Cap Strips 300 strips PN 4323032 7500 Fast system MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 200 plates PN 4366932 20 plates PN 4346906 MicroAmp Optical Adhesive Film PN 4311971 7900HT Fast system MicroAmp Optical 96 Well Reaction Plate with Barcode standard 96 well block 500 plates PN 4326659 20 plates PN 4306737 e MicroAmp Optical Adhesive Film PN 4311971 e MicroAmp Optical Film Compression Pad PN 4312639 for use with one plate e MicroAmp Snap On Optical Film Compression Pad PN 4333292 for use with automation accessory 7900HT Fast system MicroAmp Fast Optical 96 Well Reaction Plate with Barcode Fast 96 well block 200 plates PN 4366932 20 plates PN 4346906 MicroAmp Optical Adhesive Film PN 4311971 MicroAmp Optical Film Compression Pad PN 4312639 for use with one plate e MicroAmp Snap On Optical Film Compression Pad PN 4333292 for use with automation accessory 7900HT Fast system e MicroAmp Optical 384 Well Reaction Plate with Barcode 384 well block 1000 plates PN 4343814 500 plates PN 4326270 50 plates PN 4309849 e MicroAmp Optical 384 Well Reaction Plate 1000 plates PN 4343370 e MicroAmp Optical Adhesive Film PN 4311971 7500 system 48 TagMan Universal Master Mix Il Protocol Appendix B Ordering Information Real time PCR s
10. IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical n CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury N DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations MSDSs The MSDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining MSDSs see MSDSs on page 63 IMPORTANT For the MSDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer TaqMan Universal Master Mix Il Protocol Preface How to use this guide How to use this guide Text conventions This guide uses the following conventions e Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu
11. PCR Step 5 3 jj PCR amplification of cDNA Forward Primer 5 3 5 Cycle 2 GR1312b Reverse Primer Figure 3 Two step RT PCR TagMan Universal Master Mix Il Protocol 57 Appendix D Chemistry Overview About two step RT PCR About TaqMan MGB Probes About AmpliTaq Gold DNA Polymerase UP Ultra Pure About uracil N glycosylase About ROX passive reference 58 The TaqMan MGB probes contain A reporter dye for example FAM dye linked to the 5 end of the probe A minor groove binder MGB at the 3 end of the probe MGBs increase the melting temperature T without increasing probe length Afonina et al 1997 Kutyavin et al 1997 they also allow for the design of shorter probes A nonfluorescent quencher NFQ at the 3 end of the probe Because the quencher does not fluoresce Applied Biosystems real time PCR systems can measure reporter dye contributions more accurately The AmpliTaq Gold DNA Polymerase UP Ultra Pure enzyme is identical to AmpliTaq Gold DNA Polymerase but the enzyme is further purified through a proprietary process to reduce bacterial DNA introduced from the host organism The purification process ensures that non specific false positive DNA products due to bacterial DNA contamination are minimized during PCR When AmpliTaq Gold DNA Polymerase is added to the reaction mixture at room temperature the inactive enzyme is not capable
12. a a rn 54 Chemistry Overview kk kk aa kk kk KK KK KK KK KK KK KK KK QR 57 About two step RT PCR adan as d a a tee a a z d 57 Safety anan 3 dawn Av pe Tabs Mota ed oe iau d is 61 General chemical safety 00 kk kk kK KK KK KIR tenes 62 MSDSS iii eke Ai IL AS A DR rn 63 Chemical waste safety 3s su sd sa alek Ed a tk el ee ees 64 Biological hazard safety ii sa a aba eh kl belal ak A la EX fe he 65 Ghemical alerts 5c the toe d a a ste anak a Mahe lu nan E ita 65 Bibliography six amatori Venet rie d a a hana Salad othe dba aa 67 Documentation xanay a KE a tae l ea AAA eS 69 Related documentation kk kk a 69 Send us your Comments x 3 yk ca ka Ka el e Ere all i hn y y la an arek Eee 70 E II la at ont ati i EP cani A ll te n da i ai atei 11 TaqMan Universal Master Mix II Protocol Preface Safety information Note For general safety information see this Preface and Appendix E Safety on page 61 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below
13. be discovered Their expression levels vary greatly among species and tissues Kim et al 2004 Low abundant miRNAs have been difficult to detect based on current technologies such as cloning Northern hybridization Lim et al 2003 microarrays and other techniques The TaqMan MicroRNA Assays use looped primer RT PCR a new real time quantification method to accurately detect mature miRNAs Each TaqMan MicroRNA assay includes One tube containing miRNA specific RT primer One tube containing a mix of miRNA specific forward PCR primer specific reverse PCR primer miRNA specific TagMan MGB probe The TaqMan MicroRNA Assays are available for a range of species Because many mature miRNA sequences are identical across related species many assays for human are also valid for mouse and rat For the most current list of assays visit the Applied Biosystems website at www appliedbiosystems com TaqMan Universal Master Mix Il Protocol 19 TaqMan Universal Master Mix II Materials and equipment Materials and equipment Reagents not supplied The reagents below are not supplied with the TaqMan Universal Master Mix II Materials and equipment Source TaqMan MicroRNA Reverse Transcription Kit 200 reactions 4366596 1000 reactions 4366597 TaqMan MicroRNA Assays are specifically optimized to work with the MuLV Reverse Transcriptase contained in the TaqMan MicroRNA Reverse Transcrip
14. is licensed by Life Technologies Inc under U S patents and foreign equivalents for research purposes only No right for use in other applications including the diagnosis of disease in humans animals or plants under any patents owned by Life Technologies Inc are covered by the purchase of this product TRADEMARKS Trademarks of Life Technologies Corporation and its affiliated companies Applied Biosystems AB Design ABI PRISM DNAZap FAM GeneAmp JOE MagMAX Megaplex MicroAmp mirVana Primer Express RecoverAll RiboPure RNA ater RNAqueous RNaseZap RNA to cDNA RNA to Cy ROX Sample to SNP StepOne StepOnePlus TAMRA TET TURBO DNA free VICE AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc TRI Reagent is a registered trademark of Molecular Research Center Inc Microsoft and Excel are registered trademarks of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4428173 Rev B 07 2010 Protocol Appendix A TaqMan Universal Master Mix Il Protocol Contents Preface Tnm V Safety information croi sadr ius kalk xl lk aha lk lk aa a lk arl kil ay a d kana abe adi Ras V How to use this guide ssir cx ks alal sk xax a a kanala eee vi How to obtain support kk kk kk kK kk KK KK KK KOK KI KK KK KK kk kk kK kk kk kk kK kk kk kk kk kk vi TaqMan
15. method and variations for analyzing the relative changes in gene expression from Real Time quantitative PCR experiments Real Time PCR Systems Chemistry Guide Chapter 3 PN 4348358 27 TaqMan Universal Master Mix II Analyze the results 28 TaqMan Universal Master Mix ll Protocol Section 3 Genotyping Analyze the results Section 3 Genotyping Purpose Use the TaqMan Universal Master Mix II to perform genotyping of single nucleotide polymorphisms SNPs The master mix can be used with a genomic DNA and any TaqMan genotyping assay including TaqMan SNP Genotyping Assays Custom TaqMan SNP Genotyping Assays TaqMan Drug Metabolism Genotyping Assays TaqMan Pre Designed Assay Reagents for Genotyping About this section This section provides information on performing PCR using TaqMan Universal Master Mix II with TagMan SNP Genotyping Assays TaqMan Drug Metabolism SNP Genotyping Assays or Custom TaqMan Probes and Sequence Detection Primers For detailed information about specific procedures outlined in this protocol consult the appropriate instrument user guide A procedural overview is also provided in the TaqMan Universal Master Mix II Quick Reference Card PN 4428174 TaqMan Universal Master Mix Il Protocol 29 TaqMan Universal Master Mix II Materials and equipment Materials and equipment Reagents not supplied The reagents below are not supplied with TaqMan Universal Master Mix II
16. plate into the thermal cycler then start the run Read the plate After PCR amplification you perform an endpoint plate read on a real time PCR instrument The system software uses the fluorescence measurements from each well made during the plate read then plots R signal values The software determines which alleles are in each sample for later genotyping analysis Refer to the genotyping section of the appropriate instrument documentation for instructions on how to use the system software to perform the plate read and analysis Analyze the results The system software records the results of the genotyping run on a scatter plot of Allele 1 versus Allele 2 Each well of the 96 well or 384 well reaction plate is represented as an individual point on the plot for example see Figure 2 2 3 0 3 0 1 0 3 0 5 0 7 0 9 1 1 1 3 1 5 Figure 2 The clusters in the plot show the three genotypes of one SNP 34 TaqMan Universal Master Mix ll Protocol Appendix A Troubleshooting This appendix divides the troubleshooting information according to application Match your symptom with one of the observations below Find the Possible cause then follow the Recommended action Gene expression quantitation experiments Amplification curve shows abnormal plot and or low AR values 36 m Amplification curve shows arising baseline o ooo oooo 36 Multicomponent signal for ROX dye
17. preventing further amplification Since fluorescent contaminants may interfere with this assay and give false positive results it may be necessary to include a No Amplification Control tube that contains sample but no enzyme If the absolute fluorescence of the No Amplification Control is greater than that of the No Template Control after PCR fluorescent contaminants may be present in the sample or in the heat block of the thermal cycler TaqMan Universal Master Mix Il Protocol 55 Appendix C PCR Good Laboratory Practices Preventing contamination 56 TaqMan Universal Master Mix ll Protocol Appendix D Chemistry Overview About two step RT PCR Gene quantitation assays using TaqMan Universal Master Mix II and TaqMan Gene Expression Assays are performed in a two step RT PCR 1 In the reverse transcription RT step cDNA is reverse transcribed from RNA 2 Inthe PCR step PCR products are quantitatively synthesized from cDNA samples using the TaqMan Universal Master Mix II The figure below illustrates two step PCR Note Figure 3 does not show hybridization of the TaqMan MGB probe See Figure 5 on page 59 for details on how the TaqMan MGB probe is used in the PCR step Extension of primer on mRNA 5 3 mRNA RT lt 5 cDNA Random Step Primer Synthesis of 1st cDNA strand i J Extension of primer on cDNA 3 5 CDNA 5 Forward Primer Completion of 2nd cDNA strand A Cycle 1
18. single stranded cDNA from total RNA samples using the TaqMan MicroRNA Reverse Transcription Kit TaqMan MicroRNA Assays are specifically optimized to work with the MuLV Reverse Transcriptase contained in the TagMan MicroRNA Reverse Transcription Kit Applied Biosystems cannot guarantee assay performance if you use other reverse transcriptase enzymes For optimal performance of the TagMan MicroRNA Reverse Transcription Kit and of TaqMan MicroRNA Assays Applied Biosystems recommends using RNA with the following characteristics Free of inhibitors of reverse transcription and PCR e Dissolved in PCR compatible buffer Free of RNase activity Nondenatured Do not denature the RNA Denaturation of the RNA may reduce the yield of cDNA for some miRNA targets Use 1 to 10 ng of total RNA per 15 uL RT reaction Note Prepare RT master mix using the TaqMan MicroRNA Reverse Transcription Kit components before preparing the reaction 1 Allow the kit components to thaw on ice 2 Ina polypropylene tube prepare the RT master mix by scaling the volumes below to the desired number of reactions Applied Biosystems recommends preparing 110 of the required volume to account for pipetting error This procedure assumes that you are quantifying miRNA from a single total RNA sample Component Volume HL per 15 uL reaction 100 mM dNTPs with dTTP 0 15 MultiScribe Reverse Transcriptase 50 U uL 1 00 10X Reverse Transcr
19. software to set the baseline and threshold for the amplification curves either automatically or manually The baseline refers to the initial cycles of PCR in which there is slight change in fluorescence signal The intersection of the threshold with the amplification plot defines the C in real time PCR assays The threshold is set above the background and within the exponential growth phase of the amplification curve See pages 13 and 15 for information on setting the threshold and baseline Using the comparative C4 method you can use endogenous controls to normalize the expression levels of target genes by correcting differences in the amount of cDNA loaded into PCR reactions To normalize human total RNA samples an appropriate constitutively expressed endogenous control must be selected Common mRNA control transcripts are available as TagMan Endogenous Controls but must be validated for the individual researcher s samples More information about TaqMan Endogenous controls is available on the Applied Biosystems Web site Refer to the following documents for more information about analyzing your data Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide PN 4351684 Applied Biosystems 7300 7500 7500 Fast Real Time PCR Systems Absolute Quantification Getting Started Guide PN 4347825 e Livak and Schmittgen 2001 Provides the derivation assumptions and applications of the 2 A4Ct
20. 0 Time mm ss 2 00 10 00 00 15 1 00 t Required for optimal UNG activity not needed when UNG is not present in the reaction The 10 minute 95 C step is required to activate the AmpliTaq Gold UP enzyme Run Mode Standard Default Sample Volume 20 uL Auto Increment Settings Accept default values default is 0 Ramp Rate Settings Accept default values default is Standard Data Collection Accept default values default is 60 C Run the plate Refer to the appropriate instrument user guide for detailed instructions on loading and running the PCR plates see Related documentation on page 69 To run the plate 1 In the real time PCR system software open the experiment or plate document that corresponds to the reaction plate 2 Load the reaction plate into the instrument 3 Start the run 26 TagMan Universal Master Mix Il Protocol Section 2 MicroRNA quantitation Analyze the results Analyze the results Baseline and threshold values Tools for analyzing TaqMan MicroRNA Assay results Resources for data analysis TaqMan Universal Master Mix Il Protocol Refer to the appropriate instrument documentation for instructions on how to analyze your data The general process for analyzing the data from gene expression assays involves 1 Viewing the amplification plots 2 Setting the baseline and threshold values When using a real time PCR system you can use the
21. 1 Prepare a PCR reaction mix Volume HL per sample Reaction component 50 uL 20 uL Final conc rxns rxns TaqMan Universal Master Mix Il 25 0 10 0 1x Forward primer 5 0 2 0 Optimal Reverse primer 5 0 2 0 Optimal TagMan probe 5 0 2 0 50 to 250 nM DNA sample 5 0 2 0 10 to 100 ng Water 5 0 2 0 Total 50 0 20 0 2 For single probe assays determine the optimal probe concentration by running four replicates at each 50 nM interval from 50 to 250 nM Note Use the forward and reverse primer concentrations determined in Determining optimal primer concentration on page 16 3 Run the plate on the real time PCR system using the following conditions UDG Enzyme incubation activation POR Step Cycle 40 cycles HOLD HOLD Denature Anneal extend Temp C 50 C 95 C 95 C 60 C Time mm ss 2 00 10 00 00 15 1 00 Volume pL 20 or 50 t The 2 minute 50 C step is required for optimal UNG enzyme activity The 10 minute 95 C step is required to activate the AmpliTaq Gold UP enzyme Select appropriate volume for reaction plate 4 Tabulate the results for Cz Choose the minimum probe concentrations that yield the minimum Cr Recommended sample For routine assays that are optimized as described here perform real time input for real time quantitative PCR using quantitative PCR 0 1 ng to 1 ug of DNA The determined optimum probe and
22. 300 50 300 ji 50 300 50 300 90000 J 900 300 u U u u u u C ji 50 900 50 900 50 900 50 900 300 900 300 900 300 900 300 900 900 900 900 900 900 300 J 900 900 T n Ju T n ju T n lu T 1 U Place the plate in the Applied Biosystems Real Time PCR System and follow the thermal cycling conditions UDG Enzyme incubation activation PCR Step Cycle 40 cycles HOLD HOLD Denature _ Anneal extend Temp C 50 95 95 60 Time mm ss 2 00 10 00 0 15 1 00 Volume uL 20 or 50 t Select appropriate volume for reaction plate IMPORTANT The 2 minute 50 C step is required for optimal UNG enzyme activity The 10 minute 95 C step 1s required to activate the AmpliTaq Gold UP enzyme At the end of runs tabulate the results for AR Choose the minimum forward and reverse primer concentrations that yield the maximum AR The purpose of this procedure is to determine the minimum probe concentrations that give the minimum C for each probe target Most TaqMan assays are designed and run following Applied Biosystems assay development guidelines A concentration of 900 nM primers and a 250 nM probe provides for a highly reproducible and sensitive assay TaqMan Universal Master Mix Il Protocol 17 TaqMan Universal Master Mix II Using TaqMan Universal Master Mix Il with custom TaqMan probes and primers To determine the optimal probe concentration
23. 4267 Tris EDTA TE buffer 10 mM Tris HCI 1 mM EDTA pH 8 0 made using AM9849 DNase free RNase free sterile filtered water DNAZap Solution two 250 mL bottles AM9890 RT PCR Grade Water ten 1 75 mL bottles AM9935 DNase free water AM9914G See Optional user supplied reagents for gene expression quantitation on page 51 for a list of optional user supplied reagents See Real time PCR systems PCR systems and consumables on page 48 for a list of compatible real time PCR system consumables See Consumables and equipment on page 52 for a list of required laboratory consumables and equipment TaqMan Universal Master Mix II Protocol Section 1 Gene expression quantitation Workflow Workflow The following figure shows the process for performing gene expression experiments Perform the reverse transcription RT reaction Prepare the RT reaction mix Prepare the RT reaction plate y Run the RT reaction plate Perform the PCR Prepare the PCR reaction plate y Run the PCR reaction plate Analyze the results View the amplification plots for the entire plate y Set the baseline and threshold values to determine the threshold cycles C for the amplification curves Use the relative standard curve method or the comparative C method to analyze the data TaqMan Universal Master Mix Il Protocol 7 TaqMan Universal Master Mix II Perform reverse transcription Perform rev
24. 7 confidence level threshold cycle C 72 The R value of an unreacted sample This value may be obtained from the early cycles of a real time run those cycles prior to a detectable increase in fluorescence This value may also be obtained from a reaction not containing template A result with a low probability 0 3 of resulting from chance The PCR cycle number at which the fluorescence meets the threshold in the amplification plot TaqMan Universal Master Mix II Protocol Part Number 4428173 Rev B 07 2010 International Sales Applied Headquarters Bi t 5791 Van Allen Way Carlsbad CA 92008 For our office locations please call the division osys ems Phone 650 638 5800 Toll Free 800 345 5224 headquarters or refer to our Web site at www appliedbiosystems com www appliedbiosystems com about offices cfm
25. 9 Appendix B Ordering Information Gene expression assays and arrays products Gene expression assays and arrays products The following gene expression products are available from Applied Biosystems For a complete list of assays and arrays go to www appliedbiosystems com Assay or array For more information TaqMan Express Plates www allgenes com TaqMan MicroRNA Assays miRNA appliedbiosystems com Custom TaqMan Small RNA Assays Contact Applied Biosystems Sales Custom TaqMan Probes and Primers www appliedbiosystems com TaqMar Arrays TaqMan Custom Arrays TaqMan Gene Signature Array TaqMan Gene Sets taqmanarray appliedbiosystems com Megaplex Pools for microRNA Expression Analysis Megaplex RT Primers Megaplex PreAmp Primers e TaqMan MicroRNA Arrays miRNA appliedbiosystems com TaqMan Gene Expression Assays dried in MicroAmp Optical 96 Well Reaction Plates Probes and primers synthesized by Applied Biosystems to your sequence and choice of quencher and reporter dyes Reverse transcription kits and reagents To obtain cDNA from RNA samples Applied Biosystems recommends the reverse transcription kits listed in the following table For a complete list of kits and reagents go to www appliedbiosystems com Kit Source High Capacity RNA to cDNA Kit 50 rxns PN 4387406 Applied Biosystems TaqMan RNA to C
26. Primer Express Software Version 3 0 Getting Started Guide primers PN 4362460 Determining optimal The purpose of this procedure is to determine the minimum primer concentrations primer concentration giving the maximum AR The Applied Biosystems Real Time PCR Systems can provide additional data for optimization using the minimum threshold cycle C Refer to your instrument user manual for more information To determine the optimal primer concentration 1 Prepare a PCR reaction mix for primer optimization Volume pL per sample Reaction component 50 uL 20 uL Final conc rxns rxns TagMan Universal Master Mix Il 25 0 10 0 1x Forward primer 5 0 2 0 50 to 900 nM Reverse primer 5 0 2 0 50 to 900 nM TaqMan probe 2 5 uM 5 0 2 0 250 nM DNA sample 5 0 2 0 10 to 100 ng Water 5 0 2 0 Total 50 0 20 0 _ 16 TaqMan Universal Master Mix ll Protocol Section 1 Gene expression quantitation Using TaqMan Universal Master Mix II with custom TaqMan probes and primers 2 Run at least four replicates of each of the nine conditions as shown Forward primer nM 50 300 900 50 50 50 300 50 900 50 primer nM 300 50 300 300 300 900 300 900 50 900 300 900 900 900 3 Load the plate with four replicates of each condition as shown 5 Determining optimal probe concentration 5 6 T 11 300 50 300 50 300 50 900 50 900 50 m Im u U tu B j 50
27. SNP Kit Protocol 4402136 Applied Biosystems TaqMan Sample to SNP Quick 4402745 Reference Card Real Time PCR Systems Chemistry Guide 4348358 TaqMan SNP Genotyping Assays Protocol 4332856 Note For additional documentation see How to obtain support on page vi 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 IMPORTANT The e mail address above is for submitting comments and suggestions relating only to documentation To order documents download PDF files or for help with a technical question see How to obtain support on page vi 70 TaqMan Universal Master Mix Il Protocol AR value s fold difference full replicate minimum fold difference multicomponenting normalization replicate technical or PCR R n Glossary The difference between the R value and the R value It reliably indicates the magnitude of the signal generated by the given set of PCR conditions The following equation expresses the relationship of these terms AR Rat R where R Emission Intensity of Reporter PCR with template Emission Intensity of Passive Reference R Emission Intensity of Reporter PCR without template or early Emission Intensity of Passive Reference eyeles ot a Teak time reactiona See threshold cycle C The measured rat
28. TaqMan Universal Master Mix II with TagMan Gene Expression Assays Custom TaqMan Gene Expression Assays or Custom TaqMan Probes and Sequence Detection Primers For detailed information about specific procedures outlined in this protocol consult the appropriate instrument user guide A procedural overview is also provided in the TaqMan Universal Master Mix II Quick Reference Card PN 4428174 TaqMan Universal Master Mix Il Protocol TaqMan Universal Master Mix II Materials and equipment Materials and equipment Reagents not supplied Optional user supplied reagents Plastics not supplied Consumables and equipment not supplied The reagents below are not supplied with the TaqMan Universal Master Mix II Materials ao High Capacity RNA to cDNA Kit 50 rxns 4387406 High Capacity RNA to 500 reactions 4390779 aisla ME 200 reactions 4390778 50 reactions 4390777 15 reactions 4390776 High Capacity RNA to 500 reactions 4390713 CDNA Master Mix with No 200 reactions 4390712 50 reactions 4390711 15 reactions 4390710 RNase inhibitor N8080119 TaqMan Gene Expression Assays inventoried 4331182 TaqMan Gene Expression Assays made to order 4351372 Custom TaqMan Gene Small scale 20X 144 x 50 uL rxns 4331348 PAPUE SION ABSANS Medium scale 20X 300 x 50 uL rxns 4332078 Large scale 60X 1160 x 50 uL rxns 4332079 TagMan PreAmp Master Mix Kit 40 rxns 438
29. Universal Master Mix II KK eens 1 Product information cies genders a khan Ad ad a ee AE De ee ae alee a wea a il da eS 1 Materials and equipment kk kk kk 00 kk kK kk kK KK KK KK kK KK KK kk kk kk kK kk kk kk kk kk 2 Before you De il x 3 xx a dilan kk kak bala a 078 a KA a a X W4 3 Section 1 Gene expression quantitation 0 000 eee ee K KK KA 5 Materials and equipment kk kk kk kk kk kK kk kK KK KK ee 6 eise w O__rr P eeeoeiebeebbbbnn 7 Perform reverse transcription i kk kk aa 8 Perform real time PCR amplification kk kk kk kK KK KK KK KK KK K KI eee 9 Analyze the results eee eee 13 Using TaqMan Universal Master Mix Il with custom TaqMan probes and primers 16 Section 2 MicroRNA quantitation c ne 19 Materials and equipment kk kk kK KK KK KK KK KK kk KK KK KK kk kk kk KK kk kk ka 20 NS E bb 21 Perform reverse transcription kk kk kK KK KK KK KK KIR KK K KK KK KK KK KK KK KK ka 22 Perform real time PCR amplification kk kk KK KK KK KK KK KK ee 24 Analyze the results skari aly a a ais RR 27 Section 3 Genotyping s re srini kd c ee eee eee eee 29 Materials and equipment kk kK kK kK kK KK KK KK KK KK KK KK kk kk kk kk kk kk ka 30 Welal DB yp yrwrwsrs e mozr e a 31 Before YOU DEJA kall kwan k n ka a eser Nw a n 31 Perform Genotyping xa a ka su hk eee ae aa a a ala
30. affect the fluorescent probes Prior to use mix the TaqMan Universal PCR Master Mix thoroughly by swirling the bottle Prepare the PCR reaction mix before transferring to the reaction plate for thermal cycling and fluorescence analysis PCR reaction Applied Biosystems recommends performing four PCR replicates per RT components reaction The recommended reaction volume is 20 uL Prepare the plate so that each PCR reaction contains the components as listed in the following table Component 204i ection TaqMan MicroRNA Assay 20X 1 00 Product from RT reaction Minimum 1 15 Dilution 1 33 TaqMan Universal Master Mix Il no UNG 10 00 Nuclease free water 7 67 Total Volume 20 t Foroptimal performance of TaqMan MicroRNA Assays Applied Biosystems strongly recommends that you use Applied Biosystems TaqMan Universal Master Mix Il No UNG 24 TaqMan Universal Master Mix II Protocol Section 2 MicroRNA quantitation Perform real time PCR amplification Prepare the PCR Note The following procedure assumes that you are testing one individual assay reaction plate 1 Scale the volumes listed below to the appropriate number of reactions Applied Biosystems recommends including four replicates per reaction Prepare on ice Reagent Volume uL per 20 pL reaction TaqMan Universal Master Mix Il no UNG 10 00 Nuclease free water E y if 767 Total Volume i 17 67 2 Mix g
31. all assays or in an unusually large number of assays One or more of the reaction components was not added Verify that the cDNA TaqMan Gene Expression Assay and TaqMan Universal Master Mix Il were added to the reaction plate If the master mix is missing the passive reference fails Incorrect dye components were selected Check the dye components settings and reanalyze the data The annealing temperature on the thermal cycler was too high for the primers and or probe Verify that the thermal cycler is set to the correct annealing and extension temperatures Ensure that the thermal cycler is calibrated and maintained regularly Inappropriate reaction conditions were used Troubleshoot the RT PCR optimization Degraded template Determine the quality of the template e Rerun the assay with fresh template e Use RNase free reagents e Use an RNase inhibitor Inhibitors present in the reaction Verify the presence of an inhibitor 1 Create a serial dilution of your sample 2 Run the serial dilution with an expressing assay for example an endogenous control If an inhibitor is present low concentrations yield higher than expected C values High concentration means more inhibition because the sample is not diluted 3 Rerun the assay with purified template The baseline and or threshold was improperly set Refer to your real time PCR system user guide for procedures on settin
32. amplification with any of the instruments and compatible plates listed in Appendix B Ordering Information on page 47 IMPORTANT TaqMan Universal Master Mix II is not supported for use with Fast Mode thermal cycling conditions When using TaqMan Universal Master Mix II on the StepOne StepOnePlus 7500 Fast or 7900HT Fast instruments use Standard mode thermal cycling conditions If you use assays other than the TaqMan assays or use thermal cycling conditions other than those specified in this protocol validate your assays and re optimize your thermal cycling conditions as needed Refer to Real Time PCR Systems Chemistry Guide PN 4348358 for more information on selecting thermal cycling conditions TaqMan Universal Master Mix Il Protocol 3 TaqMan Universal Master Mix II Before you begin 4 TaqMan Universal Master Mix II Protocol Section 1 Gene expression quantitation Before you begin Section 1 Gene expression quantitation Purpose Use TaqMan Universal Master Mix II with the DNA target of your choice including cDNA or plasmid DNA You can use TaqMan Universal Master Mix II with any TaqMan assay or quantitative PCR application such as e Pathogen detection Copy number analysis Microarray validation Differential gene expression analysis Viral load quantitation MicroRNA quantitation see Section 2 on page 19 About this section This section provides a protocol for performing PCR using
33. aster mix Item Volume n roe S Nut r TagMan Universal Mini Pack 1 x 1 mL tube 40 4440043 pcs SM 1 Pack 1 x 5 mL bottle 200 4440040 2 Pack 2 x 5 mL bottles 400 4440047 5 Pack 5 x 5 mL bottles 1000 4440048 10 Pack 10 x 5 mL bottles 2000 4440049 Bulk Pack 1 x 50 mL bottle 2000 4440041 TagMan Universal Mini Pack 1 x 1 mL tube 40 4440042 MI Pack 1 x 5 mL bottle 200 4440038 2 Pack 2 x 5 mL bottles 400 4440044 5 Pack 5 x 5 mL bottles 1000 4440045 10 Pack 10 x 5 mL bottles 2000 4440046 Bulk Pack 1 x 50 mL bottle 2000 4440039 TaqMan Universal Master Mix II Protocol Compatible real time instruments Before you begin Prevent contamination Select an instrument and reaction plate Fast reagents and thermal cycling conditions TaqMan Universal Master Mix II Before you begin The TaqMan Universal Master Mix II may be used for real time or plate read endpoint detection of DNA or cDNA Analysis is performed using any of the following real time PCR systems StepOne or StepOnePlus Real Time PCR System Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Applied Biosystems 7900HT 7900HT Fast Real Time PCR System Applied Biosystems 7700 Sequence Detection System Applied Biosystems 7000 Sequence Detection System Review Appendix C PCR Good Laboratory Practices on page 53 IMPORTANT Use TaqMan Universal Master Mix II with Standard mode thermal cycling conditions only You can perform PCR
34. ation Follow established PCR good laboratory practices Shifting R value during the early cycles of the PCR cycles 0 to 5 Fluorescence did not stabilize to the buffer conditions of the reaction mix Note This condition does not affect PCR or the final results e Reset the lower value of the baseline range e Use automatic baselining TaqMan Universal Master Mix Il Protocol 41 Appendix A Troubleshooting Troubleshooting gene expression experiments Table 5 Troubleshooting gene expression experiments continued Observation Possible cause Recommendation Noisy signal above the threshold Evaporation Check the seal of the optical adhesive cover for leaks Empty well due to inaccurate pipetting Check the calibration of the pipettes Pipette more than 5 uL of sample Well is labeled with a detector assay in the plate document experiment but the well is empty Be sure your plate document experiment is set up correctly Exclude the well and reanalyze the data 42 TaqMan Universal Master Mix II Protocol Appendix A Troubleshooting Troubleshooting genotyping experiments Troubleshooting genotyping experiments Observation 1 No or low amplification x Wenn 09 14 19 Possible cause Recommendation Samples Sample degradation Run an agarose gel to verify that DNA is degraded Incorrect DNA qua
35. bine the components shown in Table 3 a Determine the reaction volume appropriate to the instrument and plate see Table 4 b Multiply the volume for one reaction component see Table 4 by the total number of reactions c Add the volume calculated from step 4b for each component to the tube Table 3 PCR reaction mix volume uL well Volume pL per reaction Component 5 pL rxn 10 uLrxn 25 pL rxn TagMan Universal Master Mix II 2X 2 50 5 0 12 50 TaqMan genotyping assay mix 20X 0 25 0 5 1 25 DNase free water 1 25 2 5 6 25 Total 4 0 8 0 20 0 t For ease of use dilute 40X and 80X Assay Mixes to 20X working solutions with 1X TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 Use DNase free water Table 4 Recommended volumes according to instrument System Plate well volume Reaction volume per well 7900HT 384 block 384 wells 0 02 mL 5 to 20 uL 7300 7500 7900HT 96 wells 0 2 mL 20 to 50 uL 7500 Fast 7900HT Fast 96 wells 0 1 mL 10 to 30 uL StepOne StepOnePlus 48 wells 0 1 mL 10 to 30 uL 5 Cap the tube s briefly vortex to mix the solutions then briefly centrifuge them to spin down the contents and to eliminate air bubbles 32 TaqMan Universal Master Mix II Protocol 10 11 Section 3 Genotyping Perform genotyping Into each well of a reaction plate pipette the PCR reaction mix volume 4 8 or 20 uL appropriate to your plate seal the
36. bleshooting Troubleshooting gene expression experiments Table 5 Troubleshooting gene expression experiments continued Observation Possible cause Recommendation Multicomponent signal for ROX dye is not linear Pure dye components spectra are incorrect Rerun the pure dye spectra Incorrect dye components were selected Select the correct dyes for the data analysis Amplification curve shows weak amplification Sequence mismatches between target and assay sequences Perform bioinformatics For more information refer to the Custom TaqMan Genomics Assays Protocol Submission Guidelines PN 4367671 e Bioinformatic Evaluation of a Sequence for Custom TaqMan Gene Expression Assays Tutorial from www appliedbiosystems com Degraded reagents and or probe Check the expiration date of the reagents e Verify that you follow the correct handling and storage conditions e Avoid excessive freeze thaw cycles Consider diluting the 60X TaqMan Gene Expression Assay to a 20X working stock Degraded or contaminated template e Improve the sample integrity extraction methods See Perform reverse transcription on page 8 Check each template preparation by agarose gel electrophoresis or bioanalyzer to determine the Purity only one product should be formed Level of degradation e Use RNase free sterile filtered water Inhibitors present in the reaction e V
37. clusters 0 0 ee KK KK KK 45 m Observation 5 Chicken feet clusters 00 c RR KK KK 46 TagMan Universal Master Mix Il Protocol 35 Appendix A Troubleshooting Troubleshooting gene expression experiments Troubleshooting gene expression experiments Table 5 Troubleshooting gene expression experiments Observation Possible cause Recommendation Amplification curve shows abnormal plot and or low AR values Linear view Amplification Plot ee Log view Amplification Plot The baseline was set improperly some samples have Cr values lower than the baseline stop value Refer to your real time PCR system user guide for procedures on setting the baseline Switch from manual to automatic baselining or move the baseline stop value to a lower C4 2 cycles before the amplification curve for the sample crosses the threshold Log view corrected Amplification Plot An amplification signal is detected in the early cycles no baseline can be set because the signal is detected too early Dilute the sample to increase the C value Amplification curve shows a rising baseline Linear view e Li Primer and probe interaction Adjust the threshold manually e Select another assay from the same gene 36 TaqMan Universal Master Mix II Protocol Appendix A Trou
38. e for instructions on how to configure the plate plate document or experiment See Related documentation on page 69 for a list of user documentation for Applied Biosystems real time PCR systems When creating plate documents experiments use the following parameters Thermal Cycling Parameters UNG Polymerase incubation activation POR System Cycle 40 cycles Hold Hold Denature Anneal extend Temp C 50 95 95 60 Time mm ss 2 00 10 00 00 15 1 00 Volume pL 20 or 50 t Required for optimal UNG activity omit if UNG is not present in the reaction The 10 minute 95 C step is required to activate the AmpliTaq Gold UP enzyme IMPORTANT Omit the 2 minute 50 C step if you are using TaqMan Universal Master Mix II no UNG Run Mode 9600 emulation default Sample Volume Plate format Reaction volume MicroAmp Optical 96 Well Reaction Plate 50 uL MicroAmp Fast Optical 96 Well Reaction Plate e MicroAmp Optical 384 Well Reaction Plate 20 uL e MicroAmp Fast Optical 48 Well Reaction Plate Auto Increment Settings Accept default values default is 0 Data Collection Accept default values default is 60 C Ramp Rate Settings Accept default values default is Standard TaqMan Universal Master Mix II is not supported for use with Fast Mode thermal cycling conditions When using TaqMan Universal Master Mix II on the StepOne StepOnePlus
39. e of the samples in other assays to rule out problems caused by contamination or degradation Instrument One marker is assigned to multiple Ensure that you use only one marker per assay assays TaqMan Universal Master Mix Il Protocol 45 Appendix A Troubleshooting Troubleshooting genotyping experiments Observation 5 Chicken feet clusters Possible cause Recommendation Samples Incorrect DNA quantitation Perform concentration measurements PCR inhibitors Dilute the DNA sample Variable sample input Check the performance of the samples in other assays Requantitate the DNA if applicable or ensure that the sample input for DNA extraction is within the recommended range Reagents Reagents expired or mishandled Perform the assay again with newly prepared reagents Ensure that the reagents are stored correctly Reagents not added to the well Visually inspect the well Evaporation Ensure that the reaction plate is sealed properly If recommended use a compression pad ROX dye is not in the master mix Use TaqMan Universal Master Mix Il or TagMan Genotyping Master Mix Insufficient mixing of reagents Ensure that the reagents are mixed properly then rerun the reaction Instrument Thermal cycler is poorly calibrated Check the thermal cycling conditions and make sure that the thermal cycler is correctly calibrated ROX dye is not selected Ensure that the proper
40. eamplification reactions Instrument Wrong reporter dyes chosen Verify the dye settings and reanalyze the plate read ROX dye is not selected Ensure that the proper passive reference is selected Observation 3 Clusters too close 40 13 18 23 28 33 Possible cause Recommendation Samples Sample degradation Run an agarose gel to verify if DNA is degraded Reagents Probe degradation Perform the assay again with newly prepared reagents Ensure that the reagents are stored correctly Assay design Verify that the probe designs are within good T range Instrument Too many cycles run If the reaction has been thermal cycling for more than 40 cycles rerun the PCR with fewer cycles 44 TaqMan Universal Master Mix II Protocol Observation 4 Too many clusters Appendix A Troubleshooting Troubleshooting genotyping experiments 40 Possible cause Recommendation Genetics The probe sequence may contain a Check the SNP database to see if an additional SNP has been second SNP discovered Copy number There are more than Perform a copy number assay to determine the copy number two copies of the SNP Perform comparative sequencing SNP is multi allelic Perform comparative sequencing to verify the presence of more than two alleles Repeat the experiment to verify that the performance is consistent Samples Sample contamination Check the performanc
41. ease Process for research is obtained by the purchase of i both Licensed Probe and Authorized 5 Nuclease Core Kit ii a Licensed 5 Nuclease Kit or iii license rights from Applied Biosystems The use of this product is covered by U S patent claims and patent claims outside the U S The purchase of this product includes a limited non transfer able immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research The right to use this product in the 5 Nuclease Process under the applicable claims of U S patents and patent claims outside the United States can be obtained through purchase of an Authorized 5 Nuclease Core Kit Except under separate license rights available from Applied Biosystems no right under any other patent claim or to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other com mercial consideration or to sublicense repackage with other products or resell in any form is conveyed expressly by implication or by estoppel This product is for research use only Human diagnostics uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 U S A The use of uracil N glycosylase for carryover prevention
42. eference dye Precipitation in the TaqMan buffers Besure to mix the tubes well Use TaqMan Universal Master Mix II Be sure to mix thoroughly to produce a homogenous solution Degraded TaqMan buffers Verify that the kits have been stored according to the instructions on the packaging and have not expired R on R vs Cycle plot is very high ROX dye was not selected as the passive reference when the plate document experiment was set up Select the ROX dye as the passive reference then reanalyze the data Small AR PCR efficiency is poor Recheck the concentration of the reagents Quantity of starting target is low low copy number of target Increase the quantity of the starting target TaqMan Universal Master Mix Il Protocol 39 Appendix A Troubleshooting Troubleshooting gene expression experiments Table 5 Troubleshooting gene expression experiments continued Observation Possible cause Recommendation No template control NTC shows amplification Contaminated reagents contaminated with gDNA amplicon or plasmid clones e Rerun the assay using new reagents e Be sure your workspace and equipment are cleaned properly e Use UNG e Run no reverse transcription controls to rule out genomic DNA contamination e gDNA contamination only Design an assay that spans an exon exon boundary Standard curve has a poor slope or poor c
43. emical waste safety Chemical waste hazards Chemical waste safety guidelines 64 n CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles To minimize the hazards of chemical waste e Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasse
44. ently Centrifuge to bring solution to the bottom of the tube 3 Add 17 67 uL of the PCR master mix water mixture per 20 uL PCR reaction into a polypropylene tube the PCR reaction tube 4 Transfer 1 0 uL of 20X TaqMan MicroRNA Assay mix labeled Real Time into the PCR Reaction tube 5 Transfer 1 33 uL of the RT product from the RT reaction tube into the PCR reaction tube 6 Mix gently Centrifuge to bring solution to the bottom of the plate 7 Prepare the PCR reaction plate by dispensing 20 uL of the complete PCR master mix including primer and RT product into each of four wells 8 Seal the plate with an optical adhesive cover then centrifuge the plate briefly to spin down the contents and eliminate any air bubbles 9 Apply a compression pad to the plate if required by your real time PCR system TaqMan Universal Master Mix Il Protocol 25 TaqMan Universal Master Mix II Perform real time PCR amplification Set up the experiment Refer to your instrument documentation for instructions on how to configure the or plate document plate document experiment See Related documentation on page 69 for a list of documentation for Applied Biosystems real time PCR systems When creating plate documents experiments use the following parameters Thermal Cycling Parameters UNG Polymerase incubationt activation POR System Cycle 40 cycles Hold Hold Denature Anneal extend Temp C 50 95 95 6
45. er 3 uL of RT primer tube labeled RT Primer from each assay set into the corresponding RT reaction tube or plate well 6 Seal the tube and mix gently Centrifuge to bring solution to the bottom of the tube 7 Incubate the tube on ice for 5 minutes and keep on ice until you are ready to load the thermal cycler Run the microRNA RT 1 Leaving the thermal cycler in the 9600 Emulation mode default use the reaction plate following parameter values to program the thermal cycler Step type Time min Temperature C HOLD 30 16 HOLD 30 42 HOLD 5 85 HOLD co 4 2 Set the reaction volume to 15 0 uL 3 Load the reaction tube or plate into the thermal cycler 4 Start the reverse transcription run TaqMan Universal Master Mix Il Protocol 23 TaqMan Universal Master Mix II Perform real time PCR amplification Perform real time PCR amplification During the target amplification step the AmpliTaq Gold DNA polymerase amplifies target cDNA synthesized from the RNA sample using sequence specific primers from the TaqMan Assay Plates You must perform the PCR step on a Real Time PCR system Traditional thermal cyclers cannot be used because they cannot detect and record the fluorescent signals generated by the cleavage of TaqMan probes Reagent preparation Keep all TagMan MicroRNA Assays protected from light in the freezer guidelines until you are ready to use them Excessive exposure to light may
46. erase chain reaction PCR You can use TaqMan Universal Master Mix II to amplify complementary DNA cDNA and DNA targets for a variety of applications including quantitation and genotyping The mix is available with or without UNG About this protocol This protocol describes the two primary applications of the TaqMan Universal Master Mix II quantitative RT PCR and genotyping Although TaqMan Universal Master Mix II can be used in a broad variety of PCR applications this document describes the use of the master mix with pre optimized TaqMan assays Because analysis methods vary greatly between applications this protocol provides general guidelines for the analysis of data generated from experiments that use TaqMan Universal Master Mix II and TaqMan assays For detailed information about data analysis or the procedures outlined in this protocol consult your the appropriate documentation for your instrument see Related documentation on page 69 About the kit TaqMan Universal Master Mix II has been optimized for use with primers and TaqMan probes that have been designed according to Applied Biosystems guidelines The master mix can be used with custom TaqMan assays available from the Applied Biosystems custom assay service or with pre optimized assays such as TaqMan Gene Expression Assays TaqMan MicroRNA Assays TaqMan Drug Metabolism Genotyping Assays TaqMan SNP Genotyping Assays For RNA quantitati
47. erify the presence of an inhibitor a Create a serial dilution of your sample b Run the serial dilution with an expressing assay for example an endogenous control If an inhibitor is present low concentrations yield higher than expected C values High concentration means more inhibition because the sample is not diluted c Rerun the assay with purified template e Improve sample integrity extraction methods See Perform reverse transcription on page 8 Poor reverse transcription RT conversion to cDNA e Check the RNA sample for degradation e Input RNA could be too concentrated or too dilute Verify the concentration by optical density OD make new serial dilutions of template RNA from original stock then repeat the RT PCR e Ensure that the RT PCR setup is performed under the appropriate conditions to avoid premature cDNA synthesis Check the RT reagents for contamination and or degradation Amplification curve shows low ROX dye passive reference dye Inaccurate pippetting Little or no TaqMan Universal Master Mix Il Follow accurate pipetting practices TaqMan Universal Master Mix Il Protocol 37 Appendix A Troubleshooting Troubleshooting gene expression experiments Table 5 Troubleshooting gene expression experiments continued Observation Possible cause Recommendation Amplification curve shows no amplification of the sample C 40 across
48. ersal Master Mix II Protocol 4428173 TaqMan Universal Master Mix Il Quick Reference Card 4428174 Table 7 Resources for gene expression quantitation experiments System Document ae Applied Biosystems 7900HT Fast Real Absolute Quantification Getting Started Guide 4364014 ana Syston land SDSSEISIBISO Relative Quantification Getting Started Guide 4364016 User Bulletin Performing Fast Gene Quantification 4352533 Applied Biosystems 7300 7500 7500 Fast Absolute Quantification Getting Started Guide 4347825 pes Ime RER SYSTE Relative Quantification Getting Started Guide 4347824 Getting Started Guide for Standard Curve Experiments 4387779 Getting Started Guide for Comparative C j Relative Standard 4387783 Curve Experiments Applied Biosystems StepOne or Getting Started Guide for Standard Curve Experiments 4376784 StepOnePlus Real Time PCR System Getting Started Guide for Comparative C Relative Standard 4376785 Curve Experiments All Real Time PCR Systems Chemistry Guide 4348358 TaqMan Gene Expression Assays Protocol 4333458 TaqMan Gene Expression Assays Quick Reference Card 4401212 Table 8 Resources for microRNA quantitation experiments System Document cei Applied Biosystems 7900HT Fast Real User Guide 4351684 Time PCR System and SDS Enterprise Database Applied Biosystems 7300 7500 7500 Fast Absolute Quantification Getting Started Guide 4347825 Real Time P R System Getting Started Guide for Standard Curve Experiments 4387779 Applied Biosyste
49. erse transcription Synthesis of single stranded cDNA from RNA is the first step in the RT PCR process which requires you to 1 Prepare the reverse transcription RT reaction mix 2 Prepare the RT reaction plate 3 Perform reverse transcription To obtain cDNA from RNA samples Applied Biosystems recommends using a Applied Biosystems reverse transcription kit See to Reverse transcription kits and reagents on page 50 for a list of recommended products For additional RT guidelines and instructions refer to the appropriate protocol You can download the protocols for Applied Biosystems kits at http docs appliedbiosystems com search taf RNA template For optimal performance Applied Biosystems recommends using RNA that is uidelines RE Eu g Free of inhibitors of reverse transcription and PCR Dissolved in TE buffer or PCR compatible buffer Free of RNase activity Reagent and sample To ensure optimal performance reparation guidelines a T prep g Use nuclease free pipet tips and reagents to minimize degradation of the RNA Observe standard laboratory practices when handling RNA 8 TaqMan Universal Master Mix II Protocol Section 1 Gene expression quantitation Perform real time PCR amplification Perform real time PCR amplification PCR reagent handling and preparation Determine the number of required reactions TaqMan Universal Master Mix Il Protocol Target amplification using cDNA as t
50. g the baseline and threshold e Switch from automatic to manual baselining or from manual to automatic e Lower the threshold value to within the appropriate range Assay design or synthesis failure The wrong sequence was submitted to Applied Biosystems e Verify that the sequence that you submitted is correct Check for an alternative transcript or a splice variant Assay is designed in a variable region of the gene transcript Verify that the location targeted by the assay is not within the 5 untranslated region UTR which can be highly variable between transcripts If the assay is designed within the 5 UTR select a different assay that is within the coding region of the transcript Otherwise select an assay for an alternative transcript or splice variant cDNA conversion failed Check the RNA integrity and concentration Check for RNase activity e Follow Applied Biosystems recommended thermal profile e Repeat the RT step using new reagents 38 TaqMan Universal Master Mix II Protocol Appendix A Troubleshooting Troubleshooting gene expression experiments Table 5 Troubleshooting gene expression experiments continued Observation Possible cause Recommendation Amplification curve shows samples within the same assay that have differently shaped curves The baseline was set improperly Refer to your real time PCR system user guide for procedures on se
51. hawed resuspend the samples by briefly vortexing and then centrifuge the tubes Determine the number of reactions to perform for each assay Applied Biosystems recommends performing four replicates of each reaction Include extra reactions approximately 11096 of the required volume to compensate for the volume loss that occurs during reagent transfers For example if using a 96 well plate prepare enough reaction mix for approximately 106 reactions Be sure to include on each plate A gene expression assay for each cDNA sample Endogenous control assays Optional No template controls NTCs for each assay on the plate IMPORTANT You can run multiple assays on one reaction plate Include controls for each assay that you run on a plate TaqMan Universal Master Mix II Perform real time PCR amplification Prepare the PCR 1 Prepare the reaction mix for each sample using the components listed below reaction plate Volume pL per reaction Component Final conc 50 uL 20 uL rxns rxns TaqMan Universal Master Mix II 2X 25 0 10 0 1X TaqMan Gene Expression Assay 20X 2 5 1 0 1x cDNA template H O 22 5 9 0 1 to 100 ng Total Volume 50 0 20 0 See www allgenes com for TaqMan Gene Expression Assay information Use 10 to 100 ng of cDNA plus RNase free water Calculate the volume of each component of the PCR reaction mix by multiplying the volume of each component by the nu
52. he TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites Figure 5 When the probe is intact Figure 5 and Figure 6 the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer F rster 1948 Lakowicz 1983 Forward TaqMan i a Primer MGB probe ceke 5 3 3 5 5 3 Reverse Primer Figure 5 Polymerization Forward TaqMan N a Primer MGB probe due 5 y 3 5 5 3 p 5 Reverse Primer Figure 6 Strand displacement The DNA polymerase cleaves only probes that are hybridized to the target Figure 7 Cleavage separates the reporter dye from the quencher dye the separation of the reporter dye from the quencher dye results in increased fluorescence by the reporter The increase in fluorescence occurs only if the target sequence is complementary to the probe and is amplified during PCR Because of these requirements nonspecific amplification is not detected TaqMan Universal Master Mix Il Protocol 59 Appendix D Chemistry Overview About two step RT PCR Forward ale TaqMan y Primer P MGB probe g g PO 5 5 3 P 5 Reverse Primer Figure 7 Cleavage Polymerization of the strand continues but because the 3 end of the probe is blocked no extension of the probe occurs during PCR Figure 8 TaqMan MGB probe
53. he template is the second step in the RT PCR process In this step the DNA polymerase from the TaqMan Universal Master Mix II amplifies target cDNA synthesized from the RNA sample using sequence specific primers and a TaqMan probe for example a probe from the TaqMan Gene Expression Assay mix IMPORTANT You must perform the PCR step on a real time PCR system Traditional thermal cyclers cannot be used because they cannot detect and record the fluorescent signals generated by the cleavage of TaqMan probes Note If you choose to use Custom TaqMan Probes and Sequence Detection Primers rather than a TaqMan Gene Expression Assay or a Custom TaqMan Gene Expression Assay see Using TaqMan Universal Master Mix II with custom TaqMan probes and primers on page 16 for more information Following these guidelines ensures optimal PCR performance Keep the TaqMan assays in the freezer away from light until you are ready to use them Excessive exposure to light may affect the fluorescent probes Prior to use Mix the TaqMan Universal Master Mix II thoroughly by swirling the bottle Thaw frozen TaqMan assays by placing them on ice When thawed resuspend the samples by vortexing then centrifuge the tubes briefly Resuspend the TaqMan reagents for example the TaqMan Gene Expression Assay mix by vortexing and then centrifuge the tube briefly Thaw frozen samples by placing them on ice When t
54. hemical manufacturer Before handling any chemicals refer to the MSDS provided by the manufacturer and observe all relevant precautions 52 TagMan Universal Master Mix ll Protocol Appendix C PCR Good Laboratory Practices Sample preparation When preparing samples for PCR amplification Use a positive displacement pipette or aerosol resistant pipette tips Follow proper pipette dispensing techniques to prevent aerosols Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation 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 Centrifuge tubes before opening Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Clean lab benches and equipment periodically with 10 bleach solution Use DNA Zap Solution PN AM9890 TaqMan Universal Master Mix Il Protocol 53 Appendix C PCR Good Laboratory Practices Preventing contamination Preventing contamination False positives 54 PCR assays require special laboratory practices to avoid false positive amplifications Kwok and Higuchi 1989 The high throughput and repe
55. io of the quantity of template in Sample A over the quantity of template in Sample B where quantity A gt quantity B so that the ratio is gt 1 Repeated wells of the same sample with the same assay where the contents of each well go through all experimental steps sample preparation reverse transcription and PCR separately The smallest fold difference that is statistically significant The term used to distinguish the contribution each individual dye makes to the fluorescent spectra The overlapping spectra from the dye components generate the composite spectrum which represents one reading from one well The Passive Reference 1 a dye included in the 10X TaqMan Buffer A does not participate in the 5 nuclease PCR The Passive Reference provides an internal reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescent fluctuations due to changes in concentration or volume Identical reactions that contain identical components and volumes and evaluate the same sample The R value of a reaction containing all components including the template Normalization is accomplished by dividing the emission intensity of the reporter dye by the emission intensity of the Passive Reference to obtain a ratio defined as the R normalized reporter for a given reaction tube TaqMan Universal Master Mix Il Protocol 71 Glossary statistically significant to a 99
56. ion it is important to keep the annealing temperatures greater than 55 C and to refrigerate PCR products at 2 to 8 C in order to prevent amplicon degradation Use primers that contain dA nucleotides near the 3 ends so that any primer dimer generated is efficiently degraded by UNG at least as well as any dU containing PCR products The further a dA nucleotide is from the 3 end the more likely that partially degraded primer dimer molecules may serve as templates for a subsequent PCR amplification Production of primer dimer could lower the amplification yield of the desired target region If primers cannot be selected with dA nucleotides near the ends the use of primers with 3 terminal dU nucleotides should be considered Single stranded DNA with terminal dU nucleotides are not substrates for UNG Delort et al 1985 and thus the primers will not be degraded Biotin dUMP derivatives are not substrates for UNG The concentration of UNG and the time of the incubation step necessary to prevent amplification of contaminating dU containing PCR product depends on the PCR conditions necessary to amplify your particular DNA sequence and the level of contamination expected In most cases using UNG at 1 U 100 mL reaction and incubation at 50 C for two minutes is sufficient Do not attempt to use UNG in subsequent amplification of dU containing PCR template such as in nested PCR protocols The UNG will degrade the dU containing PCR product
57. iption Buffer 1 50 RNase Inhibitor 20U uL 0 19 Nuclease free water 4 16 Total 7 00 t Each 15 uL RT reaction consists of 7 uL master mix 3 uL primer and 5 uL RNA sample 3 Mix gently Centrifuge to bring solution to the bottom of the tube 4 Place the RT master mix on ice until you prepare the microRNA reaction TaqMan Universal Master Mix II Protocol Section 2 MicroRNA quantitation Perform reverse transcription Prepare the microRNA 1 For each 15 uL RT reaction combine RT master mix from step 2 on RT reaction page 22 with total RNA in the ratio of 7 uL RT master mix 5 uL total RNA For example combine 7 7 uL of RT master mix with 5 5 uL of total RNA Remember to include the same proportion of excess volume of total RNA that you did for the RT master mix In this example a 1096 excess volume was included for both RT master mix and total RNA Applied Biosystems recommends that you use 1 to 10 ng of total RNA per reaction 2 Mix gently Centrifuge to bring the solution to the bottom of the tube Do not exceed 2000 RPM or 5 minutes when centrifuging 3 Before opening the RT Primer tubes thaw the tubes on ice and mix by vortexing then centrifuge them 4 For each 15 uL RT reaction dispense 12 0 uL of RT master mix containing total RNA from step 1 into a 0 2 mL polypropylene reaction tube This is the RT reaction tube Alternatively you can dispense into a single well of a 96 well reaction plate 5 Transf
58. is not linear 37 m Amplification curve shows weak amplification 37 Amplification curve shows low ROX dye passive reference dye 37 m Amplification curve shows no amplification of the sample C4 40 across all assays or in an unusually large number of assayS o 38 Amplification curve shows samples within the same assay that have differently shaped curves 0 2 0 0 KK KK KK KK KK KK KK KK KK KK KK KK 39 Amplification curve shows no amplification of the sample C4 40 in the target assay codes obs got O dnin a noha boned adhe 39 m Decrease in ROX dye fluorescence passive reference dye 39 B R onR vs Cycle plot is very high KK KK KK KK KK KK KK KK 39 E Sal AR lk bke d l di bek an klan tt to e te ee i e 39 m No template control NTC shows amplificati0M 40 m Standard curve has a poor slope or poor correlation coefficient 40 m Endogenous control Cys vary or do not normalize the sample well 40 m Simultaneous increase in fluorescence from both the passive reference ROX dye and the reporter dye s lees 40 Genotyping experiments B Observation 1 No or low amplificati0N o oo oooooooo oo 43 B Observation 2 No clusters 02 0 KK KK KK KK KK KK KK KK KK KK KK 44 m Observation 3 Clusters too close 2 AWK KK KK KK KK KK KK KK KK KK 44 m Observation 4 Too many
59. ix for each dilution point on the curve then transfer to the reaction plate Endogenous control Cs vary or do not normalize the sample well Endogenous control is not consistently expressed across the samples Selecting another endogenous control Sample concentrations vary widely If desired quantitate and normalize samples before running them Inaccurate pipetting Check the calibration of the pipettes e Pipette more than 5 uL of sample Simultaneous increase in fluorescence from both the passive reference ROX dye and the reporter dye s Evaporation Check the seal of the optical adhesive cover for leaks 40 TaqMan Universal Master Mix II Protocol Appendix A Troubleshooting Troubleshooting gene expression experiments Table 5 Troubleshooting gene expression experiments continued Observation Possible cause Recommendation High standard deviation of replicates inconsistent data Cr varies Inefficient mixing of reagents e Increase the length of time that you mix the reagents e Make a master mix for each dilution point on the curve then transfer to the reaction plate e Validate your mixing process by running a replicate plate Inaccurate pipetting Check the calibration of the pipettes e Pipette more than 5 uL of sample Threshold was set improperly Set the threshold above the noise and where the replicates are tightest Refer to your
60. l ala W A ee lk ee 32 Analyze theresults i nece ae xeu neka eet Rh Rok enc e Reka ea aide d dag EH RARE DA ELA ate 34 Troubleshooting kk kk kk kk kK KK kK kK KK KK KK KK KK KK e 35 Troubleshooting gene expression experiments kk kk kK KK KK KK ees 36 Troubleshooting genotyping experiments kk kk kk kk KK eee 43 Observation 1 No or low amplification kk kk kk kk KK KK KK KI KK eee 43 Observation 2 No clusters 4k kk kk kk kk kK kk kK KK KK KK KK KK KK KK KK KK KK KK KK kk a 44 Observation 3 Clusters too close kk kk kk kK kK KK KK KK KK KI KIR KI KIR KK KK KI KK KK ka 44 Observation 4 Too many clusters kk kk ne 45 Observation 5 Chicken feet clusters kk kk kk kK kK KK RR eee 46 Contents Appendix B Appendix C Appendix D Appendix E Ordering Informati n iris 47 How to order rca a Ree bl ead Sew KR ee en QNA ees a ee dee i 47 Real time PCR systems PCR systems and consumables ocooccccccc 48 Gene expression assays and arrays products 00 00 kk kK KK eee 50 Reverse transcription kits and reagents cca 50 Optional user supplied reagents for gene expression quantitation 51 Consumables and equipment 00 kk kk kk kk KK tee 52 PCR Good Laboratory Practices ZEKI KK 53 Sample preparation 0 0 0 0 KIR KK KI KK KK KK kk kK kk kk lk lk kk kk kk kk kk 53 Preventing contamination sisesed 0 0 0 44 a lih l aha lb da dra la
61. mber of replicates for each sample Applied Biosystems recommends performing four replicates of each reaction Select the reaction size depending on the reaction plate used Prepare 11096 of the required volume to account for pipetting error Usel to 100 ng of cDNA per replicate IMPORTANT For optimal performance of TaqMan Gene Expression Assays use 1 to 100 ng of cDNA per 20 or 50 uL reaction 2 Cap the tube s 3 Vortex the tube s briefly to mix the solutions 4 Centrifuge the tube s briefly to spin down the contents and eliminate any air bubbles from the solutions 5 Transfer the appropriate volume of each reaction mixture to each well of an optical plate as specified in the following table Plate format Reaction volume MicroAmp Optical 96 Well Reaction Plate 50 uL MicroAmp Fast Optical 96 Well Reaction Plate e MicroAmp Optical 384 Well Reaction Plate 20 uL e MicroAmp Fast Optical 48 Well Reaction Plate 6 Cover the plate with a MicroAmp Optical Adhesive Film For standard 96 well plates you may use MicroAmp Optical Caps 7T Centrifuge the plate briefly to spin down the contents and eliminate air bubbles from the solutions 8 Apply a compression pad to the plate if required by your real time PCR system 10 TagMan Universal Master Mix Il Protocol Section 1 Gene expression quantitation Perform real time PCR amplification Run the PCR reaction Refer to your instrument user guid
62. ms StepOne or Getting Started Guide for Standard Curve Experiments 4376784 StepOnePlus Real Time PCR System All Real Time PCR Systems Chemistry Guide 4348358 TaqMan MicroRNA Reverse Transcription Kit Protocol 4367038 TaqMan Universal Master Mix Il Protocol 69 Documentation Send us your comments Table 9 Resources for genotyping experiments Part System Document number Applied Biosystems 7300 7500 7500 Fast Allelic Discrimination Getting Started Guide 4347822 REAL TINE PEN val n Getting Started Guide for Genotyping Experiments 4387784 Applied Biosystems 7900HT Fast Real User Guide 4351684 Time PCR System and SDS Enterprise Database GeneAmp PCR System 9700 GeneAmp PCR System 9700 Base Module User Guide 4303481 GeneAmp PCR System 9700 96 Well Sample Block Module 4316011 User Guide GeneAmp PCR System 9700 Dual 384 Well Sample Block 4304215 Module User Guide GeneAmp PCR System 9700 0 5 mL Sample Block Module 4307808 User Guide GeneAmp PCR System 9700 Auto Lid Dual 96 Sample 4343363 Block Module and Dual 96 Well Sample Block Module User Guide GeneAmp PCR System 9700 Auto Lid Dual 384 Sample 4310838 Block Module User Guide Applied Biosystems StepOne and Getting Started Guide for Genotyping Experiments 4376786 StepOnePlus Real Time PCR Systems Applied Biosystems Veriti Thermal Cycler User Guide 4375799 All Applied Biosystems TaqMan Sample to
63. n curves c Using the relative standard curve method or the comparative C method to analyze the data Data analysis varies depending on the real time PCR system that you use See Related documentation on page 69 for a list of applicable documents When using a real time PCR system you can use the software to set the baseline and threshold for the amplification curves either automatically or manually The baseline refers to the initial cycles of PCR in which there is slight change in fluorescence signal The intersection of the threshold with the amplification plot defines the C in real time PCR assays The threshold is set above the background and within the exponential growth phase of the amplification curve The system software calculates baseline and threshold values for a detector based on the assumption that the data exhibit the typical amplification curve shown in Figure Experimental error such as contamination or pipetting errors can produce atypical data that can cause the software algorithm to generate incorrect baseline and threshold values for the associated detector IMPORTANT After an analysis verify that the baseline and threshold were called correctly for each well by viewing the resulting amplification plots and adjust the values manually if necessary a Plateau phase a b Linear phase lb c Exponential geometric Threshold e phase N d Background e Baseline d 5
64. nous control such as GAPDH to account for variability in the initial concentration and quality of the total RNA and in the conversion efficiency of the reverse transcription reaction All amplicons in these determinations should follow the amplicon design criteria around the Primer Express Software Refer to the Real Time PCR Systems Chemistry Guide PN 4348358 for additional information about relative quantitation TaqMan Universal Master Mix Il Protocol 15 TaqMan Universal Master Mix II Using TaqMan Universal Master Mix Il with custom TaqMan probes and primers Using TaqMan Universal Master Mix Il with custom TaqMan probes and primers To design custom probes and primers for a real time quantitative PCR assay Determine your target template and amplicon Design your custom sequence detection primers and TaqMan probe s Determine the optimal concentrations of the sequence detection primer and custom TaqMan probe s Perform real time quantitative PCR Determining target A target template is a DNA sequence including a cDNA genomic DNA or template and amplicon plasmid nucleotide sequence Design primers to amplify amplicons short segments of DNA within the target sequence The shortest amplicons work the best Consistent results are obtained for amplicon size ranges from 50 to 150 bp Designing custom Primers and probes can be designed using Primer Express Software as described TaqMan probes and in the
65. ntitation genomic Perform concentration measurements only PCR inhibitors Dilute the DNA sample Too much or too little starting Titrate sample input for the DNA extraction step material Too little DNA was used for PCR Perform another 10 PCR cycles increase the DNA input for PCR or perform preamplification reactions Reagents Reagents expired or mishandled Perform the assay again with newly prepared reagents Ensure that storage conditions are correct Reagents not added to a well Visually inspect the well Evaporation Ensure that the reaction plate is sealed properly Use a compression pad if recommended Bubbles in the wells Ensure that the reaction plate is centrifuged before thermal cycling SNP is embedded in primer designs Perform BLAST to verify that no SNP is in the primer region If necessary redesign the primer to avoid the SNP region Instrument Wrong reporter dyes were chosen Verify the dye settings and reanalyze the plate read Thermal cycler is poorly calibrated Check thermal cycling conditions and make sure the thermal cycler is correctly calibrated TaqMan Universal Master Mix Il Protocol 43 Appendix A Troubleshooting Troubleshooting genotyping experiments Observation 2 No clusters xx Possible cause Recommendation Samples PCR inhibitors Dilute the DNA sample Too little DNA used for PCR Perform another 10 PCR cycles increase the DNA input for PCR or perform pr
66. of primer extension Any low stringency mispriming events that may have occurred will not be enzymatically extended and subsequently amplified A thermal incubation step is required for activation to ensure that active enzyme is generated only at temperatures where the DNA is fully denatured Uracil N glycosylase UNG treatment can prevent the reamplification of carryover PCR products by removing any uracil incorporated into single or double stranded amplicons Longo et al 1990 UNG prevents reamplification of carryover PCR products in an assay if all previous PCR for that assay was performed using a dUTP containing master mix See Preventing contamination on page 54 for more information about UNG The ROX Passive Reference dye provides an internal reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescent fluctuations due to changes in concentration or volume TaqMan Universal Master Mix II Protocol About the 5 nuclease assay Appendix D Chemistry Overview About two step RI PCR The 5 nuclease assay process Figure 5 through Figure 8 takes place during PCR amplification This process occurs in every cycle and does not interfere with the exponential accumulation of product Nonfluorescent quencher es Minor groove binder Reporter P Hot start DNA polymerase Figure 4 Legend for Figure 5 through Figure 8 During PCR t
67. on experiments the TaqMan Universal Master Mix II is used in the second step of a two step reverse transcription polymerase chain reaction RT PCR protocol The cDNA template used with the master mix can be generated in a reverse transcription reaction using kits available from Applied Biosystems See Reverse transcription kits and reagents on page 50 for a list of recommended products TaqMan Universal Master Mix II Protocol TaqMan Universal Master Mix II Materials and equipment Materials and equipment Storage and stability Kit components Upon receipt store the TaqMan Universal Master Mix II at 2 to 8 C Applied Biosystems does not recommend storing TaqMan Universal Master Mix II at temperatures other than 2 to 8 C or using TaqMan Universal Master Mix II after the date printed on the package and bottle label Before use thoroughly mix the TaqMan Universal Master Mix II The TaqMan Universal Master Mix II with or no UNG is supplied in a 2X concentration and contains AmpliTaq Gold DNA Polymerase UP Ultra Pure e Uracil N glycosylase UNG dNTPs with dUTP ROX Passive Reference Optimized buffer components Note TaqMan Universal Master Mix II no UNG contains all the above ingredients except UNG TaqMan Universal Master Mix II with or no UNG is supplied in a 2X concentration and is available in the following volumes M
68. orrelation coefficient Where e Poor slope a slope value of 3 32 equals approximately 10096 efficiency or Poor correlation coefficient the best correlation coefficient is 1 0 Incorrect dilutions e Redilute the samples Ensure pipettes are calibrated e Pipette more than 5 uL of sample Inaccurate pipetting Check the calibration of the pipettes e Pipette more than 5 uL of sample Inhibitors present in the reaction Verify the presence of an inhibitor 1 Create a serial dilution of your sample 2 Run the serial dilution with an expressing assay for example an endogenous control If an inhibitor is present low concentrations yield higher than expected C values High concentration means more inhibition because the sample is not diluted 3 Rerun the assay with purified template Improper reaction conditions Follow the Applied Biosystems recommended thermal cycling profile Inconsistent replicates high standard deviation Make a master mix for each dilution point on the curve then transfer to the reaction plate Range of dilution points is too narrow Increase the number of points and the logarithmic range Incorrect baseline and threshold settings Verify settings according to your real time PCR system user documentation Bad correlation coefficient only Improper mixing e Increase the length of time that you mix the reagents Make a master m
69. osal TaqMan Universal Master Mix II Protocol Appendix E Safety MSDSs MSDSs About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive anew MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you MSDSs free 24 hours a day To obtain MSDSs 1 Go to www appliedbiosystems com click Support then select MSDS 2 Inthe Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following e Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer TaqMan Universal Master Mix Il Protocol 63 Appendix E Safety Chemical waste safety Ch
70. passive reference is selected 46 TaqMan Universal Master Mix II Protocol Appendix B Ordering Information How to order The consumables and reagents in this appendix are for use with the TagMan Universal Master Mix II and can be ordered from the Applied Biosystems website This appendix contains ordering information for the following m Real time PCR systems PCR systems and consumables 48 m Gene expression assays and arrays productS oo oooooooooooo 50 m Reverse transcription kits and reagentS o ooooooooooomom ooo 50 m Optional user supplied reagents for gene expression quantitation 51 m Consumables and equipment o ooooooococorr KK KK KK KK KK KK 52 TaqMan Universal Master Mix Il Protocol 47 Appendix B Ordering Information Real time PCR systems PCR systems and consumables Real time PCR systems PCR systems and consumables The following table lists real time PCR systems thermal cyclers and consumables that can be used with TaqMan Universal Master Mix II and TaqMan assays For a complete list of PCR systems and consumables go to www appliedbiosystems com System Reaction plates and accessories 7300 system e MicroAmp Optical 96 Well Reaction Plate with Barcode 500 plates PN 4326659 20 plates PN 4306737 MicroAmp Optical Adhesive Film PN 4311971 MicroAmp Optical Film Compression Pad PN 4312639 MicroAmp Optical 8 Tube Strips 0
71. plate with a MicroAmp clear adhesive film then centrifuge the plate briefly to spin down the contents and eliminate any air bubbles Remove the clear adhesive film from the plate then pipette one control or diluted DNA sample into the appropriate well s If you use purified genomic DNA use 1 to 10 ng of genomic DNA or control DNA for each reaction in the appropriate volume see table below Volume of genomic DNA or DNA control uL PCR reaction 1 0 2 0 5 0 5 pL reaction 10 uL reaction 25 uL reaction Seal the plate using MicroAmp Optical Adhesive Film or MicroAmp Optical Caps then centrifuge the plate briefly to spin down the contents and eliminate air bubbles Apply a compression pad to the plate if required by your real time PCR system Load the plate into a real time PCR system TaqMan Universal Master Mix Il Protocol 33 TaqMan Universal Master Mix II Analyze the results Perform the PCR 1 Set up the following run conditions IMPORTANT These conditions are optimized for use only with TaqMan genotyping assays on the instruments specified in Appendix B Polymerase activation PCR System Cycle 40 cycles Hold Denature Anneal extend Temp C 95 95 60 Time mm ss 10 00 00 15 1 00 The 10 minute 95 C step is required to activate the AmpliTaq Gold UP enzyme Runspeed Standard Reaction volume 5 10 or 25 uL 2 Load the reaction
72. primer concentrations The appropriate volume of TaqMan Universal Master Mix II as described in Prepare the PCR reaction plate on page 10 The thermal cycling conditions specified in your instrument user guide 18 TaqMan Universal Master Mix Il Protocol Section 2 Purpose About this section About microRNAs About TaqMan MicroRNA Assays Available TaqMan MicroRNA Assays Section 2 MicroRNA quantitation Using TaqMan Universal Master Mix II with custom TaqMan probes and primers MicroRNA quantitation Use TaqMan Universal Master Mix II to perform microRNA quantitation using TaqMan MicroRNA Assays The TaqMan MicroRNA Assays are designed to detect and accurately quantify mature microRNAs miRNAs using Applied Biosystems real time PCR instruments This section provides a protocol for performing PCR using TaqMan Universal Master Mix II with TagMan MicroRNA assays For detailed information about specific procedures outlined in this protocol consult the appropriate instrument documentation A procedural overview is also provided in the TaqMan Universal Master Mix II Quick Reference Card PN 4428174 MicroRNAs are endogenous RNAs about 22 nucleotides long that play important regulatory roles in animals and plants by targeting mRNA transcripts for cleavage or translational repression Bartel 2004 To date hundreds of unique mature miRNAs have been identified across species with more continuing to
73. real time PCR system documentation for procedures on setting the threshold Low concentration of target Rerun the assay using more template Template absorption adhering to the tube Add a carrier for example yeast tRNA Cr value is lower than expected gDNA contamination Perform bioinformatics Design the assay to span an exon exon junction For more information refer to the Custom TaqMan Genomics Assays Protocol Submission Guidelines PN 4367671 Bioinformatic Evaluation of a Sequence for Custom TaqMan Gene Expression Assays Tutorial from www appliedbiosystems com e Verify contamination by running an RT minus reaction without the reverse transcriptase e Treat the sample with DNase More sample added than expected e Reduce the amount of sample e Quantitate and normalize the sample Template or amplicon contamination Follow established PCR good laboratory practices Amplification occurs in the no RT controls gDNA contamination e Perform bioinformatics Design the assay to span an exon exon junction For more information refer to the Custom TaqMan Genomics Assays Protocol Submission Guidelines PN 4367671 Bioinformatic Evaluation of a Sequence for Custom TaqMan Gene Expression Assays Tutorial from www appliedbiosystems com e Improve sample extraction methods to eliminate gDNA e Treat the sample with DNase Template or amplicon contamin
74. rmation Consumables and equipment Consumables and equipment The following includes required and optional laboratory equipment and materials Unless otherwise noted many items listed are available from major laboratory suppliers Materials Source Accessories for tubes of assay mixes Micronic Bvts Decapper for single caps e Decapper for eight caps e TPE cap cluster for simultaneously capping 96 individual polypropylene tubes 50 capmats bag Centrifuge with plate adapter MLS Disposable gloves MLS Microcentrifuge MLS Heat block or water bath or thermal cycler to 95 C MLS Microcentrifuge tubes 1 5 mL AM12400 Barrier Filter Tips 10 uL size Pipetman Ten 8 x 12 racks AM12640 10 pL size Eppendorf Ten 8 x 12 racks AM12635 20 uL size Ten 8 x 12 racks AM12645 1000 uL size Ten 100 ct racks AM12665 200 uL size Ten 8 x 12 racks AM12655 Pipettors Positive displacement MLS Air displacement Multichannel Vortexer MLS Microsoft Excel software or equivalent spreadsheet and analysis Software software suppliers t Other vendors supply similar products Micronic BV PO Box 604 8200 AP Lelystad Netherlands Telephone 0031 320 277 090 Fax 0031 320 277 088 United States Telephone 724 941 6411 Fax 724 941 8662 Website www micronic com Major laboratory supplier MLS For the MSDS of any chemical not distributed by Applied Biosystems contact the c
75. s TaqMan Universal Master Mix Il Protocol 65 Appendix E Safety Chemical alerts 66 TaqMan Universal Master Mix ll Protocol Bibliography F rster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Annals of Physics Leipzig 2 55 15 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lakowicz J R 1983 Principles of Fluorescence Spectroscopy ed New York Plenum Press xiv 496 pp Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 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 TaqMan Universal Master Mix Il Protocol 67 Bibliography 68 TaqMan Universal Master Mix II Protocol Related documentation Applied Biosystems documents Documentation Table 6 TaqMan Universal Master Mix Il documentation You can download the documents in Tables 6 7 8 and 9 from the Applied Biosystems Web site at http docs appliedbiosystems com search taf Document Esos TagMan Univ
76. s gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations TaqMan Universal Master Mix II Protocol Appendix E Safety Biological hazard safety Waste disposal If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious di
77. scribed here are capable of degrading or eliminating large numbers of carried over PCR products we encourage users to continue using the specific devices and suggestions described in this protocol booklet and in Kwok 1990 and Higuchi 1989 to minimize cross contamination from non dU containing PCR products or other samples TaqMan Universal Master Mix II Protocol Uracil N glycosylase UNG Prevention of PCR product carryover Fluorescent contaminants Appendix C PCR Good Laboratory Practices Preventing contamination The UNG provided in the TaqMan Universal Master Mix II is a pure nuclease free 26 kDa recombinant enzyme encoded by the Escherichia coli uracil N glycosylase gene which has been inserted into an E coli host to direct the expression of the native form of the enzyme Kwok and Higuchi 1989 UNG acts on single and double stranded dU containing DNA by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The enzyme causes the release of uracil and creates an alkali sensitive apyrimidic site in the DNA Apyrimidic sites block replication by DNA polymerases The enzyme has no activity on RNA or dT containing DNA UNG incubation at 50 C is necessary to cleave any dU containing PCR carryover products Ten minute incubation at 95 C is necessary to substantially reduce UNG activity and to denature the native DNA in the experimental sample Because UNG is not completely deactivated during the 95 C incubat
78. seases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following US Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov Chemical alerts For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page v General alerts for all Avoid contact with skin eyes and or clothing Read the MSDS and follow the chemicals handling instructions Wear appropriate protective eyewear clothing and glove
79. supplied See Real time PCR systems PCR systems and consumables on page 48 for a list of compatible real time PCR system real time PCR system consumables Consumables and _ See Consumables and equipment on page 52 for a list of required laboratory equipment not supplied consumables and equipment 30 TaqMan Universal Master Mix Il Protocol Section 3 Genotyping Workflow Workflow The following figure shows the process for performing genotyping experiments Perform the PCR Prepare the PCR mix Perform the PCR Read the plate Create and set up a plate document or experiment Perform the PCR Read the PCR reaction plate Analyze the results Review the Genotyping Plot Before you begin Quantitate the DNA For a genotyping assay add 1 to 10 ng of DNA template per reaction well To quantitate genomic DNA use a reliable method such as A260 measurements or real time quantification by RNase P If you use the RNase P method you generate a standard curve using the DNA template standards in the TaqgMan DNA Template Reagents Kit PN 401970 and the RNase P gene primers and probe in the TaqMan RNase P Detection Reagents Kit PN 4316831 For details on generating a standard curve refer to Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR at http www appliedbiosystems com support tutorials pdf quant_pcr pdf Determine the number Determine the number of reactions
80. the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles 4 WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles To minimize the hazards of chemicals e Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page 63 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disp
81. tion Kit Applied Biosystems cannot guarantee assay performance if you use other reverse transcriptase enzymes Plastics not supplied See Real time PCR systems PCR systems and consumables on page 48 for a list of compatible real time PCR system consumables Consumables and _ See Consumables and equipment on page 52 for a list of required laboratory equipment not supplied consumables and equipment 20 TaqMan Universal Master Mix Il Protocol Section 2 MicroRNA quantitation Workflow Workflow The following figure shows the process for performing miRNA quantitation Perform reverse transcription RT Prepare the miRNA RT reaction mix Prepare the miRNA RT reaction plate Run the miRNA RT reaction plate Perform real time PCR amplification Create and set up a plate document or experiment 4 Prepare the PCR reaction plate 4 Run the PCR reaction plate Analyze the results View the amplification plots for the entire plate 4 Set the baseline and threshold values to determine the threshold cycles C for the amplification curves Use the relative standard curve method or the comparative C method to analyze the data TaqMan Universal Master Mix Il Protocol 21 TaqMan Universal Master Mix II Perform reverse transcription Perform reverse transcription RNA template guidelines Per reaction input amount of total RNA Prepare the microRNA RT reaction master mix 22 Synthesize
82. tition of these assays can lead to amplification of a single DNA molecule Saiki er al 1985 Mullis and Faloona 1987 Special laboratory practices are necessary in order to avoid false positive amplifications Higuchi et al 1989 This is because of the capability for single DNA molecule amplification provided by the PCR process Saiki et al 1985 Mullis et al 1987 Saiki et al 1988 Because of the enormous amplification possible with PCR amplicon carryover can result in sample contamination Other sources of contamination could be from samples with high DNA levels or from positive control templates When dUTP replaces dTTP as a dNTP substrate in PCR and the method described below is used UNG treatment can prevent the reamplification of carryover PCR products in subsequent experiments Sninsky and Gelfand pers comm This method uses enzymatic and chemical reactions analogous to the restriction modification and excision repair systems of cells to degrade specifically PCR products from previous PCR amplifications or to degrade mis primed non specific products produced prior to specific amplifications but not degrade native nucleic acid templates The method used to make PCR products susceptible to degradation involves substituting dUTP for dTTP in the PCR mix and treating subsequent PCR mixes with the enzyme uracil N glycosylase UNG EC 3 2 2 prior to amplification Longo et al 1990 Although the protocol and reagents de
83. to perform for each assay Prepare 110 of the of required reactions required volume to account for pipetting error For example for a 96 well plate prepare enough volume for approximately 106 reactions Be sure to include on each plate at least Two no template controls NTCs Optional one genomic DNA control of known genotype IMPORTANT You can run multiple genotyping assays on one reaction plate Include controls for each assay that you run on a plate TaqMan Universal Master Mix Il Protocol 31 TaqMan Universal Master Mix II Perform genotyping Perform genotyping The first step in a genotyping assay is PCR amplification which requires you to prepare the PCR mix perform the PCR read the plate and analyze the results Prepare the PCR mix IMPORTANT Keep all TaqMan reagents protected from light until you are ready to use them Excessive exposure to light may affect the fluorescent probes Minimize freeze thaw cycles Prepare the PCR reaction mix for each assay before transferring it to the reaction plate for thermal cycling and fluorescence analysis 1 Thoroughly mix the TaqMan Universal Master Mix II by swirling the bottle 2 Thaw the frozen TaqMan assays by placing them on ice Vortex then centrifuge the tubes briefly 3 Thaw any frozen genomic DNA by placing them on ice After the samples thaw mix them if needed by vortexing then centrifuge the tubes briefly 4 In an appropriate tube com
84. tting the baseline e Switch from automatic to manual baselining or from manual to automatic e Increase the upper or lower value of the baseline range Sample quality is poor 1 Perform a quality check on the sample 2 If necessary reextract the sample Imprecise pipetting different concentrations Follow accurate pippetting practices Contamination Be sure your workspace and equipment are properly cleaned Amplification curve shows no amplification of the sample C4 40 in the target assay One or more of the reaction components was not added Check your pipetting equipment and or technique Incorrect dye components were selected Check the settings of the dye components before data analysis The gene is not expressed in the tested sample e Verify by Rerunning the sample using the same assay Running the sample using an alternative assay for the same gene Verify the known expression of the gene in the sample type Note If the recommended actions do not resolve the problem the result may be correct The reaction may not have enough copies of the target gene Verify by Rerunning the sample using the same assay Rerunning the assay using more sample Running the sample using an alternative assay for the same gene Note If the recommended actions do not resolve the problem the result may be correct Decrease in ROX dye fluorescence passive r
85. ystems PCR systems and consumables System Reaction plates and accessories 9700 instrument MicroAmp Optical 96 Well Reaction Plate with Barcode 500 plates PN 4326659 20 plates PN 4306737 ABI Prism 384 Well Clear Optical Reaction Plate with Barcode 1000 plates PN 4343814 500 plates PN 4326270 50 plates PN 4309849 MicroAmp Optical Adhesive Film PN 4311971 MicroAmp Clear Adhesive Films 100 films PN 4306311 MicroAmp Optical 8 Tube Strips 0 2 mL 1000 tubes in strips of eight PN 4316567 MicroAmp Optical 8 Cap Strips 300 strips PN 4323032 StepOne system MicroAmp Fast Optical 48 Well Reaction Plate 20 plates PN 4375816 MicroAmp 48 Well Optical Adhesive Film PN 4375323 StepOnePlus system MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 200 plates PN 4366932 20 plates PN 4346906 MicroAmp Optical Adhesive Film PN 4311971 Veriti 96 well thermal cycler MicroAmp Optical 96 Well Reaction Plate 500 plates PN 4316813 10 plates PN N8010560 MicroAmp Optical Adhesive Film PN 4311971 MicroAmp Clear Adhesive Films 100 films PN 4306311 Veriti 384 well thermal cycler MicroAmp Optical 384 Well Reaction Plate with Barcode 1000 plates PN 4343814 500 plates PN 4326270 50 plates PN 4309849 MicroAmp Optical Adhesive Film PN 4311971 TaqMan Universal Master Mix Il Protocol 4

TaqMan® Universal Master Mix II Protocol

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TaqMan&reg; Universal Master Mix II Protocol

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TaqMan® Universal Master Mix II Protocol