Fast SYBR® Green Master Mix Protocol
Contents
1. Applied Biosystems 7300 7500 7500 Fast Real Time PCR Systems Absolute Quantification Getting Started Guide PN 4347825 Real Time PCR Systems Chemistry Guide Applied Biosystems 7900HT Fast Real Time PCR Systems and 7300 7500 7500 Fast Real Time PCR Systems PN 4348358 Fast SYBR Green Master Mix Protocol Detect Nonspecific Amplification Detect Nonspecific Amplification Dissociation Curves Because SYBR Green I dye detects any double stranded DNA check for nonspecific product formation by using dissociation curve or gel analysis A dissociation curve is a graph that displays dissociation data from the amplicons of quantitative PCR runs Change in fluorescence due to a dye or probe interacting with double stranded DNA is plotted against temperature When to Generate Dissociation Curves IMPORTANT Because of the presence of UDG you must generate a dissociation curve immediately after the real time PCR run on any Applied Biosystems Real Time PCR System An Example The dissociation curves below show typical primer dimer formation The specific product is shown with a melting temperature T of 80 5 C but the primer dimer has a characteristically lower T of 15 Primer dimers are most prevalent in NTC wells and sample wells containing a low concentration of template Dissociation curve of specific product Dissociation curve of a primer dimer Temperature C Fig2. Background d Baseline e Experimental error such as contamination or inaccurate pipetting can produce data that deviate significantly from data for typical amplification curves Such atypical data cause the software algorithm to generate incorrect baseline and threshold values for the associated detector Therefore Applied Biosystems recommends reviewing all baseline and threshold values after analysis of the study data If necessary adjust the values manually as described in the appropriate instrument user manual IMPORTANT After analysis you must verify that the baseline and threshold were called correctly for each well by viewing the resulting amplification plots Fast SYBR9 Green Master Mix Protocol 3 3 Chapter3 Analyze the Data See the example amplification plots below to determine whether or not the baseline and threshold settings were correctly set Baseline Set Correctly The amplification curve begins after the maximum baseline No adjustment necessary Amplification Plot 1 000 E 1 1 000 E 4 Start End Cycle Baseline Set Too Low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value Amplification Plot Start End Cycle Baseline Set Too High The amplification curve begins before the maximum baseline Decrease the End Cycle value Amplification Plot 1 000 E 1 1
3. Major laboratory supplier MLS Disposable gloves MLS Microcentrifuge MLS NuSieve 4 3 1 agarose gels FMC BioProducts for DNA lt 1 kb PN 54928 Pipette tips with filter plugs MLS Pipettors positive displacement MLS or air displacement Polypropylene tubes MLS Tris EDTA TE Buffer pH 8 0 MLS Vortexer MLS Fast SYBR Green Master Mix Protocol Materials Required but Not Supplied Applied You can download the following documents from Biosystems http docs appliedbiosystems com search taf Documents Document S All Systems High Capacity cDNA Reverse Transcription Kit Protocol 4375575 Primer Express Software Version 3 0 Getting Started Guide 4362460 Real Time PCR Systems Chemistry Guide 4348358 StepOne and StepOnePlus Systems Applied Biosystems StepOne Real Time PCR System Getting Started Guide for Relative Standard Curve and 4376785 Comparative AAC Experiments Applied Biosystems StepOne Real Time PCR System 4376784 Getting Started Guide for Standard Curve Experiments Applied Biosystems StepOne Real Time PCR System 4376782 Installation Maintenance and Networking Guide 7500 Fast System Applied Biosystems 7300 7500 7500 Fast Real Time PCR 4347828 System Installation and Maintenance Getting Started Guide Applied Biosystems 7300 7500 7500 Fast Real Time PCR 4347824 System Relative Quantification Getti
4. 2 5 Prepare the PCR Reaction Plate 2 5 Run the PCR Reaction 2 8 Fast SYBR Green Master Mix Protocol 2 1 Chapter2 Prepare and Run the Samples Procedural Overview This diagram is an overview of the procedures for performing gene expression experiments Prepare total RNA ABI PRISM 6100 Nucleic Acid PrepStation Perform reverse transcription Create and set up a plate document SDS software c w 2 2 Prepare the PCR reaction plate Pr lt C 384 well plate 96 well 48 well O Fast plate Fast plate A s Run the PCR reaction plate PTS Real Time PCR instrument Analyze results NT Amplification Plot 2 2 Fast SYBR9 Green Master Mix Protocol Prepare the Template Prepare the Template After isolating the template examine its quality and quantity and store it properly Examine RNA Before using the Fast SYBR Green Master Mix you need to Template Quality synthesize single stranded cDNA from total RNA or mRNA samples For optimal performance Applied Biosystems recommends using RNA that is Between 0 002 and 0 2 ug uL Less than 0 005 of genomic DNA by weight Free of inhibitors of reverse transcription and PCR Dissolved in PCR compatible buffer Free of RNase activity IMPORTANT If you suspect that the RNA
5. For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste Disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply viii Fast SYBR9 Green Master Mix Protocol Biological Hazard Safety Safety ANTITE BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations
6. 8 Analyze the Data Analyze Your Results 3 2 Detect Nonspecific Amplification 3 7 Troubleshoot ED ERE EX E AS Reta es daa ete 3 9 Fast SYBR9 Green Master Mix Protocol iii Contents Appendix A Design the Assay Identify Target Sequences and Design Primers A 2 Optimize Primer Concentrations for PCR A 4 Bibliography iv Fast SYBR Green Master Mix Protocol Preface Safety Safety Safety Alert Words Fast SYBR Green Master Mix Protocol This preface contains Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N i e Indicates a potentially hazardous situation that 1f not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices N Indicates a potentially hazardous situation that 1f not avoided could result in death or serious injury N Indicates an imminently hazardous situation that if not avoided will result in death or serious injury Thi
7. 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 U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories http bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http 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 http www cdc gov Fast SYBR9 Green Master Mix Protocol ix Preface How to Obtain Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certific
8. contains RNase activity add RNase inhibitor to the reverse transcription reaction at a final concentration of 1 0 U uL Adding RNase inhibitor to the reverse transcription reaction is not necessary if the RNA is purified using the ABI PRISM 6100 Nucleic Acid PrepStation and Applied Biosystems nucleic acid purification reagents Nondenatured IMPORTANT It is not necessary to denature the RNA Denaturation of the RNA may reduce the yield of cDNA for some gene targets Examine DNA Use both of the following methods to examine DNA quality Template Quality Agarose gel electrophoresis Purified DNA should run as a single band on an agarose gel Agarose gels reveal contaminating DNAs and RNAs but not proteins Spectrophotometry The A o A go ratio should be 1 8 to 2 0 Smaller ratios usually indicate contamination by protein or organic chemicals Spectrophotometry can reveal protein contamination but not DNA or RNA contamination Quantitate the Template quantitation is critical for successful PCR reactions The Template most common way to determine DNA quantity is to measure the absorbance optical density or O D of a sample at 260 nm in a spectrophotometer Fast SYBR9 Green Master Mix Protocol 2 3 Chapter2 Prepare and Run the Samples 2 4 Store the Template One O D unit is the amount of a substance dissolved in 1 0 mL that gives an absorbance reading of 1 00 in a spectrophotometer with a 1 cm path length The
9. technology and information for life scientists Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 07 2010 amp Applied E Biosystems Part Number 4385372 Rev
10. the Fast SYBR Green Master Mix you can perform two types of quantitation relative and absolute Relative quantitation compares a target against an internal standard You can perform relative quantitation using either the standard curve method or the comparative C method Absolute quantitation compares the of an unknown sample against a standard curve with known copy numbers Relative Quantitation Method Gene expression can be measured by the quantitation of cDNA relative to a calibrator sample The calibrator sample serves as a physiological reference In a typical experiment gene expression levels are studied as a function of a treatment of cells in culture of patients or of tissue type The calibrator sample in each case is the cDNA from the untreated cells or patients or a specific tissue type All quantitations are also normalized to an endogenous 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 Refer to the following documents for more information about analyzing your data You can download the documents from http docs appliedbiosystems com search taf 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 Relative Quantification Getting Started Guide PN 4347824
11. transcribed into cDNA RT PCR 1 In the reverse transcription RT step cDNA is reverse transcribed from total RNA samples using random primers from the High Capacity cDNA Reverse Transcription Kit 2 In the PCR step PCR products are synthesized from cDNA samples using the Fast SYBR Green Master Mix Extension of primer on mRNA Bi 3 mRNA 5 a Random PH Step Oligo Primar or Synthesis of 1st cDNA strand BANAN 3 cDNA Extension of primer on cDNA 7 3 5 Forward Primer Synthesis of 2 cDNA strand PCR 5 Step 5 3 j PCR amplification of cDNA A N Forward Primer p 3 5 o o gt 2 5 O i 5 D d t Pese Figure 1 2 Two step RT PCR Fast SYBR9 Green Master Mix Protocol 1 5 Chapter 1 Introduction Contents and Storage Contents The Fast SYBR Green Master Mix is supplied in a 2X concentration Item ieu Contents Mini Pack 4385610 One 1 mL tube 100 x 20 uL reactions 1 Pack 4385612 One 5 mL tube 500 20 pL reactions 2 Pack 4385616 2x5 mL tubes 1000 x 20 uL reactions 5 Pack 4385617 5x5 mL tubes 2500 20 uL reactions 10 Pack 4385618 10x 5 mL tubes 5000 x 20 uL reactions Bulk Pack 4385614 One 50 mL tube 5000 x 20 pL reactions Storage Store the Fast SYBR Green Master Mix at 20 C Note After the first thaw Applied Biosystems recommends storing the Fast SYBR Gree
12. 000 1 000 E 1 4 1 000 E2 1 000 E3 1 000 E4 o 1 R 20 Cycle Start End Cycle 3 4 Fast SYBR Green Master Mix Protocol Analyze Your Results 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 Amplification Plot 1 000 E3 1 000 E 4 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 Amplification Plot 1 000 E 1 1 000 1 000 E 1 lt 1 000 E2 1 000 E 3 1 000 E4 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 Amplification Plot 1 000 1 000 E1 Threshold lt 1 000 E 2 1 000 E3 1 000 E4 i Fast SYBR Green Master Mix Protocol 3 5 Chapter3 Analyze the Data 3 6 Analyzing the Results Resources for Data Analysis Using
13. 5 E8 10 1 2 1 2 0 7 6 20 E9 E12 10 1 2 3 6 0 5 2 20 F1 F4 10 3 6 0 2 0 6 2 20 F5 F8 10 3 6 1 2 0 5 2 20 F9 F12 10 3 6 3 6 0 2 8 20 Fast SYBR Green Master Mix Protocol A 9 Appendix A Design the Assay A 10 Fast SYBR Green Master Mix Protocol Bibliography Ausubel F M Brent R Kingstin R E Moore D D Seidman J G Smith J A and Struhl K eds 1998 Current Protocols in Molecular Biology John Wiley and Sons New York NY p A 3 0 2 Delort A M Duplaa A M Molko D and Teoule R 1985 Excision of uracil residues in DNA mechanism of action of Escherichia coli and Micrococcus luteus uracil DNA glycosylases Nucleic Acids Res 13 319 335 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 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 Fast SYBR9 Green Master Mix Protocol Bibliography 1 Bibliography Bibliography 2 Fast SYBR Green Master Mix Protocol Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applied Biosystems is committed to providing the world s leading
14. 96 Time PCR System with Fast block Well Reaction Plates Applied Biosystems 7900HT Fast Real MicroAmp Optical 384 Well Time PCR System with 384 well block Reaction Plate Configure the For information about configuring plate documents when performing Plate Document real time quantification refer to the appropriate user guides listed in Applied Biosystems Documents on page 1 9 Prepare the PCR Reaction Plate Reminder About Before starting check that the melting point for your primers is 58 to Your Primers 60 C Refer to page A 2 for more information about identifying target sequences and designing primers Reagent Handling Follow these guidelines to ensure optimal PCR performance and Preparation Prior to use Mix the Fast SYBR Green Master Mix thoroughly by swirling the bottle Place frozen cDNA samples and primers on ice to thaw After the samples are thawed vortex them then centrifuge the tubes briefly Fast SYBR Green Master Mix Protocol 2 5 Chapter2 Prepare and Run the Samples Prepare the PCR rmi CHEMICAL HAZARD Fast SYBR Green 2 6 Reactions Master Mix 2X may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Applied Biosystems recommends performing four replicates of each reaction For the recommended reaction volume of 20 uL prepare the PCR reactions acco
15. BR Green Master Mix Protocol 1 3 Chapter 1 Introduction Chemistry Overview How the SYBR The SYBR Green I dye chemistry uses the SYBR Green I dye to Green Dye detect PCR products by binding to double stranded DNA formed Chemistry Works during PCR The process works as follows 1 When Fast SYBR Green Master Mix is added to a sample SYBR Green I dye immediately binds to all double stranded DNA 2 During the PCR AmpliTaq Fast DNA Polymerase UP amplifies the target sequence which creates the PCR product or amplicon 3 The SYBR Green I dye then binds to each new copy of double stranded DNA 4 As the PCR progresses more amplicon is created Because the SYBR Green I dye binds to all double stranded DNA the result is an increase in fluorescence intensity proportional to the amount of double stranded PCR product produced The figure below illustrates this process Step 1 The SYBR Green During PCR The SYBR Green dye within the Fast AmpliTaq Fast DNA dye then binds to SYBR Green Master Polymerase UP each new copy of Mix immediately amplifies each target double stranded binds with all double DNA stranded DNA Figure 1 1 Representation of how the SYBR Green I dye acts double stranded DNA during one extension phase of PCR 1 4 Fast SYBR Green Master Mix Protocol Chemistry Overview Using the Master When performing a two step RT PCR reaction total or mRNA must Mix in Two Step first be
16. Fast SYBR Green Master Mix Protocol KS Bee Sams Copyright 2007 2010 Applied Biosystems 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 assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PUCHASER LIMITED LICENSE Use of this product is covered by US patent claims and patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as apparatus or system claims and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This pr
17. T 8400 6 50 400 Total 167 950 measured absorbance 260 nm 0 13 sum of extinction coefficient contributions for probe 167 950 M 1cm 1 0 3 cm cuvette pathlength sum of extinction coefficient contributions x cuvette pathlength x oligonucleotide concentration 100 167 950 Mcm x 0 3 cm x C 100 258 uM Absorbance 260 nm 0 13 Fast SYBR9 Green Master Mix Protocol A 5 Appendix A Design the Assay Determine the CHEMICAL HAZARD Fast SYBR Green Optimal Primer Master Mix is a combustible liquid and vapor keep away from heat Concentration and flame It may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To optimize primer concentrations for PCR 1 Prepare a 96 well reaction plate as shown in Table A 1 on page A 9 Use 10 to 100 ng of genomic DNA or 1 to 10 ng of cDNA template The final concentration of Fast SYBR Green Master Mix is 1X Note The plate configuration accounts for four replicates of each of the following nine variations of primer concentration applied to both template and NTC wells Reverse Primier Forward Primer nM nM 50 300 900 50 50 50 300 50 900 50 300 50 300 300 300 900 300 900 50 900 300 900 900 900 2 Calibrate your instrument for SYBR Green Dye if necessary Refer to the instrument user manual for calibration ins
18. ates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities x Fast SYBR Green Master Mix Protocol Introduction Overview This chapter describes the Fast SYBR Green Master Mix and provides important information on PCR practices Purpose ofthe Kit usia ali ana Waa 1 2 Chemistry Overview senpene enira cette p es 1 4 Materials Required but Not Supplied 1 7 Prevent Contamination and Nonspecific Amplification 1 10 Fast SYBR Green Master Mix Protocol 1 1 Chapter 1 Introduction Purpose of the Kit About the Kit UDG ROX Passive Reference Fast SYBR Green Master Mix supplied in a 2X concentration is a convenient premix to perform real time PCR using SYBR Green I dye The master mix contains SYBR Green I Dye AmpliTaq Fast DNA Polymerase UP Ultra Pure Uracil DNA Glycosylase UDG dye Passive Reference dNTPs Optimized buffer components Primers template and water are the only components that you provide Fast SYBR Green Master Mix contains uracil DNA glycosylase UDG UDG is also known as uracil N glycosylase UNG UDG treatment can prevent the reamplification of carryover PCR products by remo
19. dous 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 a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain MSDSs l 2 Go to https docs appliedbiosystems com msdssearch html In the Search field of the MSDS Search page a Type in the chemical name part number or other information that you expect to appear in the MSDS of interest Fast SYBR9 Green Master Mix Protocol Safety b Select the language of your choice c Click Search 3 To view download or print the document of interest a Right click the document title b Select Open To view the document Save Target As To download a PDF version of the document to a destination that you choose Print Target To print the document 4 To have a copy of an MSDS sent by fax or e mail in the Search Results page a Select Fax or Email below the document title b Click RETRIEVE DOCUMENTS at the end of the document list c Enter the required information d Click View Deliver Selected Documents Now Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical Waste Nnm HAZARDOUS WASTE R
20. e sure that the thermal cycler mode is set to Fast see page 2 8 R vs Cycle plot is not displayed ROX dye was not selected as the passive reference when the plate document was set up Select ROX dye as the passive reference when you set up the plate document Extremely high AR or R values ROX dye was not selected as the passive reference when the plate document was set up Select ROX dye as the passive reference when you set up the plate document Evaporation Make sure that the reaction plate is sealed completely especially around the edges Lower AR values obtained in early cycles Cr value is less than 15 Adjust the upper baseline range to a value less than 15 High variability across the reaction plate ROX dye was not selected as the passive reference when the plate document was set up Select ROX dye as the passive reference when you set up the plate document Evaporation Make sure that the reaction plate is sealed completely especially around the edges High variability across replicates Reaction mix was not mixed well Mix the reaction mix gently by inversion then centrifuge briefly before aliquoting to the reaction plate Fast SYBR Green Master Mix Protocol Design the Assay Overview This appendix describes how to design primers to amplify custom target sequences for quantitation You may not have to routinely do this part of the
21. efer to Material Hazards Safety Data Sheets and local regulations for handling and disposal Norme CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Norme 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 Fast SYBR Green Master Mix Protocol vii Preface Chemical Waste minimize the hazards of chemical waste Safety Guidelines Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A 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 glasses gloves or protective clothing
22. fy the amount of cDNA template 2 Test the cDNA template for the presence of PCR inhibitors Sample degradation Prepare fresh cDNA then repeat the experiment Incorrect pipetting Prepare the reactions as described on page 2 6 Reduced number of PCR cycles in the thermal cycler protocol Increase the number of PCR cycles to the default setting of 40 see page 2 8 Primer dimer formation and residual polymerase activity 1 Prepare the reaction mixes and the reaction plate on ice 2 To ensure optimal results run the reaction plate as soon as possible after completing the reaction setup If you cannot run a reaction plate within 2 hours after completing the reaction setup store the reaction plate at 4 C Low AR or R values Extension time is too short Use the default thermal profile settings see page 2 8 Primer dimer formation and residual polymerase activity 1 Prepare the reaction mixes and the reaction plate on ice 2 To ensure optimal results run the reaction plate as soon as possible after completing the reaction setup If you cannot run a reaction plate within 2 hours after completing the reaction setup store the reaction plate at 4 C Fast SYBR9 Green Master Mix Protocol Chapter3 Analyze the Data Observation Possible Cause Action Run takes more than 40 minutes Thermal cycler mode is set to Standard or 9600 Emulation Mak
23. n Master Mix at 4 C 1 6 Fast SYBR9 Green Master Mix Protocol Materials Required but Not Supplied Materials Required but Not Supplied Plates Choose the plate appropriate for your real time instrument Instrument Platest Part Number StepOne system MicroAmp Fast Optical 48 Well Reaction Plate 20 plates 4375816 StepOnePlus MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 0 1 mL 7500 Fast 7900 HT Fast 20 Plates 4346906 systems 200 plates 4366932 MicroAmp Optical 384 Well Reaction Plate with Barcode e 50 plates 4309849 7900 HT 384 well e 500 plates 4326270 e 1000 plates 4343814 MicroAmp Optical 384 Well Reaction Plate 1000 plates 4343370 Seal all plates except StepOne system plates with MicroAmp Optical Adhesive Film PN 4360954 Seal StepOne system plates with MicroAmp 48 Well Optical Adhesive Film PN 4375323 Requires a MicroAmp Snap On Optical Film Compression Pad PN 4333292 Other Kits Item Source Applied Biosystems High Capacity cDNA Reverse Transcription Kit 200 reactions 4368814 200 reactions with RNase Inhibitor 4374966 1000 reactions 4368813 1000 reactions with RNase Inhibitor 4374967 Fast SYBR Green Master Mix Protocol 1 7 Chapter 1 1 8 Introduction Other Consumables Item Source Centrifuge with adapter for 96 well plates or Centrifuge with adapter for 384 well plates
24. nes for Designing Primers on page A 2 Do not use UDG in subsequent amplifications of dU containing PCR template such as in nested PCR protocols The UNG degrades the dU containing PCR product preventing further amplification PCR Good When preparing samples for PCR amplification Laboratory Weara clean lab coat not previously worn while handling Practices amplified PCR products or used during sample preparation and clean gloves Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Keepreactions and components capped as much as possible Usea positive displacement pipette or aerosol resistant pipette tips Clean lab benches and equipment periodically with a 10 bleach solution Fast SYBR Green Master Mix Protocol 1 11 Chapter 1 Introduction 1 12 Fast SYBR Green Master Mix Protocol Prepare and Run the Samples Overview This chapter describes how to prepare your template and samples for PCR amplification Procedural Overview 2 2 Prepare the o WA w WA He l n eee 2 3 Set Up the Plate Document
25. ng Started Guide Applied Biosystems 7300 7500 7500 Fast Real Time PCR 4347825 System Absolute Quantification Getting Started Guide 7900HT System Applied Biosystems 7900HT Fast Real Time PCR System 4351684 and SDS Enterprise Database User Guide Applied Biosystems 7900HT Fast Real Time PCR System 4352533 User Bulletin Performing Fast Gene Quantification Fast SYBR9 Green Master Mix Protocol 1 9 Chapter 1 Introduction Prevent Contamination and Nonspecific Amplification Overview Using UDG to Minimize Reamplification Carryover Products Use NAC and NTC Controls Design Primers to 1 10 Avoid Primer Dimers PCR assays require special laboratory practices to avoid false positive amplifications The high throughput and repetition of these assays can lead to amplification of a single DNA molecule Fast SYBR Green Master Mix contains uracil DNA glycosylase UDG UDG is also known as uracil N glycosylase UNG UDG treatment is useful in preventing the reamplification of carryover PCR products UDG is a 26 kDa recombinant enzyme encoded by the Escherichia coli uracil DNA glycosylase gene This gene has been inserted into E coli host to direct expression of the native form of the enzyme Kwok and Higuchi 1989 UDG acts on single and double stranded dU containing DNA It acts by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The enzyme causes the release of uracil the
26. oduct is for research use only Diagnostic 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 USA TRADEMARKS Applera Applied Biosystems AB Design ABI PRISM and Primer Express are registered trademarks and MicroAmp ROX StepOne and StepOnePlus are trademarks of Applied Biosystems or its subsidiaries in the U S and or certain other countries AmpliTaq and TaqMan are registered trademarks of Roche Molecular Systems Inc SYBR is a registered trademark of Molecular Probes Inc All other trademarks are the sole property of their respective owners Part Number 4385372 Rev C 07 2010 Contents Preface Chapter 1 Chapter 2 Chapter 3 oatety xx Re basal oe te Bey Kunkel bah Sea ane V Introduction Purpose of the Kit l l kk kk kk KK KK KK RIK KI KIR KK KK KK KK KK KK KIR 1 2 Chemistry Overview e k E eee 1 4 Materials Required but Not Supplied 1 7 Prevent Contamination and Nonspecific Amplification 1 10 Prepare and Run the Samples Procedural Overview Qada sal Ea AA UI exer EEG 2 2 Prepare the Template 2 3 Set Up the Plate Document 2 5 Run the PCR Reaction Plate 2
27. optimal results Fast SYBR9 Green Master Mix Protocol A 7 Appendix A Design the Assay Example of Nonspecific Amplification a LE 1 14 P ud 12 ail M Target Amplification 4 2 N A ti NTC nonspecific amplification 1 5 10 15 20 25 30 35 40 Cycle Number Target Amplification non specific amplification Derivative Raw Fluorescence Temperature C Figure A 1 Amplification data using SYBR Green dye chemistry a Amplification plot linear view demonstrating suspected nonspecific amplification in NTC wells b Dissociation curve analysis confirming that product in NTC wells has a melting temperature different from the specific product Fast SYBR Green Master Mix Protocol Optimize Primer Concentrations for PCR Table A 1 Plate Configuration for Primer Optimization for PCR Fast SYBR m Total Wells Green 5 pM Forward 5 uM Reverse Template Deionized Volume Master Mix Primer uL Primer uL uL Water uL uL uL A1 A4 10 0 2 0 2 2 0 7 6 20 A5 A8 10 0 2 1 2 2 0 6 6 20 A9 A12 10 0 2 3 6 2 0 4 2 20 1 4 10 1 2 0 2 2 0 6 6 20 5 8 10 1 2 1 2 2 0 5 6 20 9 12 10 1 2 3 6 2 0 3 2 20 C1 C4 10 3 6 0 2 2 0 4 2 20 C5 C8 10 3 6 1 2 2 0 3 2 20 C9 C12 10 3 6 3 6 2 0 0 8 20 01 04 10 0 2 0 2 0 9 6 20 05 08 10 0 2 1 2 0 8 6 20 09 012 10 0 2 3 6 0 6 2 20 E1 E4 10 1 2 0 2 0 8 6 20 E
28. ove Also see Select an Amplicon Site for cDNA on page A 3 RNA Design the primers as described above Fast SYBR Green Master Mix Protocol Identify Target Sequences and Design Primers Select an Selecting a good amplicon site ensures amplification of the target Amplicon Site for cDNA without co amplifying the genomic sequence pseudogenes cDNA and related genes Guidelines The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA The primer pair must be specific to the target gene the primer pair does not amplify pseudogenes or other related genes Design primers according to Primer Express Software guidelines Test the amplicons then select those that have the highest signal to noise ratio that is low C with cDNA and no amplification with no template control or genomic DNA Ifno good sequence is found you may need to examine the sequence and redesign the amplicon or to screen for more sites If the gene you are studying does not have introns then you cannot design an amplicon that amplifies the mRNA sequence without amplifying the genomic sequence In this case you may need to run RT minus controls Fast SYBR Green Master Mix Protocol A 3 Appendix A Design the Assay Optimize Primer Concentrations for PCR A 4 Overview Quantitate the Primers By independently varying the forward and reverse primer concentrations you can identify
29. procedure Identify Target Sequences and Design Primers A 2 Optimize Primer Concentrations for PCR A 4 Fast SYBR Green Master Mix Protocol A 1 Appendix Design the Assay Identify Target Sequences and Design Primers Identify Target Sequence and Amplicon Size Guidelines for Designing Primers A 2 A target template is a DNA sequence including cDNA genomic DNA or plasmid nucleotide sequence that you want to amplify Using Primer Express Software you design primers to amplify amplicons segments of DNA within the target sequence Shorter amplicons work best Consistent results are obtained for amplicon size ranges from 50 to 150 bp Using Primer Express Software Design primers using Primer Express Software as described in the Primer Express Version 3 0 Getting Started Guide PN 4362460 and Online Help General Guidelines Do not overlap primer and probe sequences The optimal primer length is 20 bases Keep the GC content in the 30 to 80 range Avoid runs of identical nucleotides If repeats are present there must be fewer than four consecutive G residues Important Keep the Tn between 58 to 60 C e Make sure the last five nucleotides at the 3 end contain no more than two G and or C bases If the template is Then DNA plasmid DNA Design the primers as described above genomic DNA cDNA Design the primers as described ab
30. process for analyzing the data from gene expression assays requires that you View the amplification plots Baseline and Usethe Sequence Detection System SDS software to automatically Threshold Values calculate or manually set the baseline and threshold for the threshold cycles for the amplification curves AAC method to analyze the results amplification curves Baseline refers to the initial cycles of PCR in which there is little Rn 3 2 change in fluorescence signal of the amplification curve Sample Threshold L eel eee ee Fe Zx III No Template Control Baseline T T T T T T T 1 0 5 10 15 20 25 30 35 40 Cycle Number Fast SYBR Green Master Mix Protocol Adjust the baseline and threshold values to determine the Use the standard curve method or the relative quantification The intersection of the threshold with the amplification plot defines the in real time PCR assays The threshold is set above the background and within the exponential growth phase Analyze Your Results View the Amplification Plots Manually Adjust the Baseline and Threshold The SDS software calculates baseline and threshold values for a detector based on the assumption that the data exhibit the typical amplification curve A typical amplification curve as shown below has a Plateau phase a Linear phase b Exponential geometric phase c
31. rding to the following procedure Using an appropriate size tube prepare the reactions for each sample Volume for One Component 20 Reaction uL Fast SYBR Green Master Mix 2X 10 0 Forward and Reverse Primerst Variable cDNA template RNase free water Variable Total Volume 20 0 t For optimal performance use a minimum of 200 nM of each primer For optimal performance use up to 20 ng of cDNA per 20 pL reaction plus RNase free water Cap the tube mix by gentle inversion then centrifuge the tube briefly to spin down the contents and eliminate any air bubbles Transfer the appropriate volume of each reaction to each well of an optical plate as specified in the table below Plate Format Volume uti MicroAmp Fast Optical 48 Well Reaction Plate 20 MicroAmp Fast Optical 96 Well Reaction Plate 20 MicroAmp Optical 384 Well Reaction Plate 20 Fast SYBR Green Master Mix Protocol Prepare the PCR Reaction Plate 4 Sealthe plate with an optical adhesive cover then centrifuge the plate briefly to spin down the contents and eliminate any air bubbles IMPORTANT Run the reaction plate within 2 hours after completing the reaction setup If you cannot do so store the plate at 4 C Fast SYBR Green Master Mix Protocol 2 7 Chapter2 Prepare and Run the Samples Run the PCR Reaction Plate Run the plate on an Applied Biosys
32. reby creating an alkali sensitive apyrimidic site in the DNA The enzyme has no activity on RNA or dT containing DNA Longo et al 1990 Because fluorescent contaminants can interfere with SYBR GreenI Dye assays and give false positive results it may be necessary to include a No Amplification Control NAC tube that contains sample but no enzyme If the absolute fluorescence of the NAC is greater than that of the No Template Control NTC after PCR fluorescent contaminants may be present in the sample or in the heat block of the thermal cycler Use primers that contain dA nucleotides near the 3 ends so that any primer dimer generated is efficiently degraded by UDG at least as well as any dU containing PCR products The farther a dA nucleotide is from the 3 end the more likely partially degraded primer dimer molecules can serve as templates for a subsequent PCR amplification Fast SYBR9 Green Master Mix Protocol Prevent Contamination and Nonspecific Amplification Production of primer dimers could lower the amplification yield of the desired target region If primers cannot be selected with dA nucleotides near the ends consider using primers with 3 terminal dU nucleotides Single stranded DNA with terminal dU nucleotides are not substrates for UDG Delort et al 1985 and therefore the primers are not degraded Biotin dUMP derivatives are not substrates for UDG For more information about designing primers see Guideli
33. s signal word is to be limited to the most extreme situations Preface Chemical Hazard vi Warning Chemical Safety Guidelines About MSDSs Obtaining MSDSs rom CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death To minimize the hazards of chemicals 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 vi 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 disposal 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 hazar
34. tems real time quantitative PCR instrument See the appropriate instrument user guide for help with programming the thermal cycling conditions or with running the plate To run the plate 1 Place the reaction plate in the instrument 2 Set the thermal cycling conditions using the default PCR thermal cycling conditions specified in the following table Select Fast Mode Instrument Step A Duration Cycles AmpliTaq Fast DNA 95 20 sec HOLD Polymerase UP Step One Activation StepOne Plus e 7500 Fast Denature 95 3 sec 40 Anneal Extend 60 30 sec AmpliTaq Fast DNA 95 20 sec HOLD Polymerase UP Activation 7900HT Fast Denature 95 1 sec 40 Anneal Extend 60 20 sec 3 Set the reaction volume according to the following table Plate Format Reaction Volume pL MicroAmp Fast Optical 48 Well Reaction Plate 20 MicroAmp Fast Optical 96 Well Reaction Plate 20 MicroAmp Optical 384 Well Reaction Plate 20 4 Start the run 2 8 Fast SYBR9 Green Master Mix Protocol Analyze the Data Overview The chapter describes how to analyze the data generated in your experiment Analyze Your Results 3 2 Detect Nonspecific Amplification 3 7 Troubleshoot 3 9 Fast SYBR Green Master Mix Protocol 3 1 Chapter3 Analyze the Data Analyze Your Results The general
35. the primer concentrations that provide optimal assay performance The primer concentrations you select should provide a low C and a high AR when run against the target template but should not produce nonspecific product formation with NTCs 1 Measure the absorbance at 260 nm of a 1 100 dilution of each primer oligonucleotide in TE buffer 2 Calculate the sum of extinction coefficient contributions for each primer extinction coefficient extinction coefficient x number of contribution bases in oligonucleotide sequence See An Example Calculation of Primer Concentration on page A 5 for an example calculation 3 Calculate the oligonucleotide concentration in uM for each primer absorbance of extinction coefficient 260 nm contribution x cuvette pathlength x concentration 100 Rearrange to solve for concentration concentration 100 absorbance 260 nm sum of extinction coefficient contribution x cuvette pathlength Fast SYBR Green Master Mix Protocol Optimize Primer Concentrations for PCR An Example Calculation of Primer Concentration In this example the concentration of a primer in TE buffer diluted 1 100 with the sequence CGTACTCGTTCGTGCTGC is calculated using the following values Number of cer Chromophore ences in Coefficient Example Contribution Sequence A 15 200 1 15 200 C 7050 6 42 300 G 12 010 5 60 050
36. tructions Note Applied Biosystems recommends that you calibrate your instrument every 6 months 3 Load the plate into an Applied Biosystems real time PCR system 4 Program the thermal cycling conditions according to the information in step 2 on page 2 8 5 Run the plate A 6 Fast SYBR Green Master Mix Protocol Confirm the Absence of Nonspecific Amplification Optimize Primer Concentrations for PCR To optimize primer concentrations for PCR continued 6 Compile the results for AR and C then select the minimum forward and reverse primer concentrations that yield the maximum AR values and low values Dissociation curves help you select the optimal primer concentrations for your SYBR quantification assays Review the linear view of the amplification plot in your NTC wells Note In Figure A 1 on page A 8 part a the strong amplification of the NTC wells indicates that significant nonspecific amplification is occurring Generate a dissociation curve on your Applied Biosystems Real Time PCR System Note In the example dissociation curve data shown in Figure A 1 on page A 8 part b the melting temperature of the product generated in the absence of template is lower than the melting temperature of the specific product generated with template This variation 1s typical of primer dimer formation and it indicates that lower primer concentration may provide
37. ure 3 1 Example of two dissociation curves Fast SYBR Green Master Mix Protocol 3 7 Chapter3 Analyze the Data Use Agarose Gels 3 8 IMPORTANT Because of the presence of UDG immediately verify to Check PCR the absence of nonspecific amplification using agarose gel Product Purity electrophoresis 1 Load 12 to 15 uL of sample well on an ethidium bromide stained 4 NuSieve 3 1 agarose gel Norme CHEMICAL HAZARD Ethidium bromide causes eye skin and respiratory tract irritation and is a known mutagen that is it can change genetic material in a living cell and has the potential to cause cancer Always use adequate ventilation such as that provided by a fume hood Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Run the gel For PCR fragments 100 bp use 80 to 100 V for 45 to 60 min For PCR fragments 100 to 250 bp use 100 to 115 V for 1 to 1 5 h Run samples 1 3 to 1 2 the length of the gel without letting the dye run off the bottom of the gel Use a UV lamp to check the migration of the samples Fast SYBR9 Green Master Mix Protocol Troubleshoot Troubleshoot Observation Possible Cause Action High C values poor precision or failed PCR reactions Insufficient cDNA template is present Use up to 20 ng of cDNA template per 20 uL reaction Quality of cDNA template is poor 1 Quanti
38. ving any uracil incorporated into single or double stranded amplicons Longo et al 1990 UDG 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 Prevent Contamination and Nonspecific Amplification on page 1 10 for more information about UDG Fast SYBR Green Master Mix contains ROX dye Passive Reference The ROX dye Passive Reference provides an internal reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescence fluctuations due to changes in concentration or volume Fast SYBR9 Green Master Mix Protocol Real Time Instruments About This Protocol Purpose of the Kit Fast SYBR Green Master Mix can be used to run experiments on the following Applied Biosystems Real Time PCR Systems Applied Biosystems StepOne Real Time PCR System Applied Biosystems StepOnePlus Real Time PCR System Applied Biosystems 7500 Fast Real Time PCR System Applied Biosystems 7900HT Fast Real Time PCR System This protocol provides Background information about Fast gene quantification assays A list of equipment and materials for using the Fast SYBR Green Master Mix Procedures for using the Fast SYBR Green Master Mix For details about specific procedures described in this protocol see Applied Biosystems Documents on page 1 9 Fast SY
39. wavelength is assumed to be 260 nm unless stated otherwise Aso values can be converted into ug uL using Beer s Law Absorbance sum of extinction coefficient contributions x cuvette 260 nm pathlength x concentration The following formulas are derived from Beer s Law Ausubel et al 1998 Concentration of single stranded DNA x 33 ug uL Concentration of double stranded DNA x 50 ug uL Concentration of single stranded RNA A x 40 ug uL Note Absorbance measurements of highly concentrated O D gt 1 0 or very dilute O D 0 05 DNA or RNA samples can be inaccurate Dilute or concentrate the DNA RNA to obtain a reading within the acceptable range Store the templates as follows Store purified RNA templates at 20 C or 70 C in RNase free water Store purified DNA templates at 20 C or 70 C in Tris HCl pH 8 0 Fast SYBR9 Green Master Mix Protocol Set Up the Plate Document Set Up the Plate Document Select a Plate for Refer to page 1 7 for part numbers of the plates PCR If you use Select Applied Biosystems StepOne Real MicroAmp Fast Optical 48 Time PCR System Well Reaction Plates Applied Biosystems StepOnePlus MicroAmp Fast Optical 96 Real Time PCR System Well Reaction Plates Applied Biosystems 7500 Fast Real MicroAmp Fast Optical 96 Time PCR System Well Reaction Plates Applied Biosystems 7900HT Fast Real MicroAmp Fast Optical
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