PowerUp™ SYBR™ Green Master Mix
Contents
1. SYBR Green Master Mix at 2 C to 8 C 8 PowerUp SYBR Green Master Mix User Guide Required Materials Plates Choose the plate appropriate for your real time instrument Instrument Plates Catalog number MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 0 1 mL e 20 plates 4346906 e 200 plates 4366932 ViiA 7 and MicroAmp Fast Optical 96 Well Reaction Plate with Barcode QuantStudio e 20 plates 4306737 systems e 500 plates 4326659 MicroAmp Optical 384 Well Reaction Plate with Barcode e 50 plates 4309849 e 500 plates 4326270 e 1000 plates 4343814 StepOne system MicroAmp Fast Optical 48 Well Reaction Plate 20 plates 4375816 StepOnePlus and MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 7500 Fast systems 0 1 mL e 20 plates 4346906 e 200 plates 4366932 7900HT and MicroAmp Optical 96 Well Reaction Plate with Barcode 7900HT Fast8 e 20 plates 4306737 systems e 500 plates 4326659 MicroAmp Optical 384 Well Reaction Plate with Barcode e 50 plates 4309849 e 500 plates 4326270 e 1000 platesv 4343814 7500 system MicroAmp Optical 96 Well Reaction Plate with Barcode e 20 plates 4306737 e 500 plates 4326659 5 Requires a MicroAmp Snap On Optical Film Compression Pad Cat no 4333292 when using the standard MicroAmp Optical 96 Well Reaction Plate Optical Seals Seal all plates except StepOne system plates with MicroAmp Optical Adhesive Fil2. G 12 010 5 60 050 T 8400 6 50 400 Total 167 950 measured absorbance at 260 nm 0 13 sum of extinction coefficient 167 950 M cm contributions for probe cuvette pathlength 0 3 cm Absorbance 260 nm sum of extinction coefficient contributions x cuvette pathlength x oligonucleotide concentration 100 0 13 167 950 Mem x 0 3 cm x C 100 C 258 uM 28 PowerUp SYBR Green Master Mix User Guide Determine the Optimal Primer Concentration Confirm the Absence of Nonspecific Amplification JN CHEMICAL HAZARD PowerUp SYBR Green Master Mix is a combustible liquid and vapor keep away from heat and flame It may cause eye skin and respiratory tract irritation Read the SDS 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 described below Use 10 to 100 ng of genomic DNA or 1 to 10 ng of cDNA template The final concentration of PowerUp 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 Primer Forward Primer nM nM 300 500 800 300 300 300 500 300 800 300 500 300 500 500 500 800 500 800 300 800 500 800 800 800 TM 2 Calibrate your instrument for SYBR Green Dye if necessary R
3. and Nonspecific Amplification ccceccceeseeeeceeeeeeeeeeeeeeeeeeeaeeeeeeeeeeeeeseeeesneeesaes 11 MOU A soasddaccensnsensasadanatadaadenndusadscesuadaeauecere 13 060 0 RRA IA 13 Prepare the Template 14 Set Up the Plate 0 0 0 7 7 7 7 77707 15 Prepare 3 ae ae a nana 15 Run the PGR Reaction Plate aii 17 Analyze Your Results 18 Detect Nonspecific Amplification oooooconcccnnccnnncccononcnnoncnnnncnnnnnnnnn cnn nn n non nn nan nn nn 22 la 8 A A A E E AA ee 24 APPO A aE E E E EE E E su cones taccdsusveectucrssceccurvestenees 26 26 Optimize Primer Concentrations for PCR ccceccccececeeceeeeceeececeeeeeceaeeeeaeeeeaeeeeeeeeeaeessaeeesaeeseneeeeneeeeeeess 28 APPOM 0 31 A nd a ee 31 Documentationsand a dd led eh ode ded o dedos teh dra ed tl da 33 PowerUp SYBR Green Mix User Guide 3 Product Information About the Reagent Hot Start UDG dUTP dTTP SYBR Green TM TM The PowerUp SYBR Green Master Mix is formulated to provide superior specificity and sensitivity It is supplied in a convenient 2X concentration premix TM to perform real time PCR using SYBR Green dye The master mix contains e SYBR Green Dye e Dual Lock DNA Polymerase with a proprietary combination of two proprietary hot start modifications for exceptional specificity gt Heat labile Uracil DNA Glycosylase
4. you can generate a dissociation curve up to 72 hours 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 Tm of 80 5 C but the primer dimer has a characteristically lower Tm of 75 C Primer dimers are most prevalent in NTC wells and sample wells containing a low concentration of template Dissociation e curve of specific product v Dissociation curve of a 2 primer dimer Temperature C Figure 3 Example of two dissociation curves 22 PowerUp SYBR Green Master Mix User Guide Optional Check Note Because of the presence of heat labile UDG you can verify the absence of PCR Product nonspecific amplification using agarose gel electrophoresis up to 72 hours after amplification Purity by Agarose Gel 1 Load 12 to 15 pL of sample per well on an ethidium bromide stained Electophoresis agarose gel made with UltraPure Agarose 1000 Cat no 16550 100 PCR Fragment Size Agarose in Agarose in TBE Buffer TAE Buffer lt 100 bp 5 6 100 250 bp 3 4 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 prov
5. 4347824 Quantification Getting Started Guide Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Absolute 4347825 Quantification Getting Started Guide 7900HT System Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise 4351684 Database User Guide Applied Biosystems 7900HT Fast Real Time PCR System User Bulletin Performing ae 4352533 Fast Gene Quantification ViiA 7 System Applied Biosystems ViiA 7 Real Time PCR System Getting Started Guides 4441434 Applied Biosystems ViiA 7 Real Time PCR System User Guide 4442661 QuantStudio 12K System Applied Biosystems QuantStudio 12K Real Time PCR System User Guide 4470050 Obtaining support For the latest services and support information for all locations go to thermofisher com lifescience At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches PowerUp SYBR Green Mix User Guide 33 Safety Data Sheets SDS Certificate of Analysis Limited warranty Safety Data Sheets SDSs are ava
6. UDG TM e ROX dye Passive Reference e dNTP blend containing dUTP dTTP e Optimized buffer components The user only needs to provide primers template and water PowerUp SYBR Green Master Mix contains the proprietary Dual Lock Taq DNA Polymerase that utilizes a combination of two hot start mechanisms to control its activity The dual hot start approach provides tight control over Tag activation preventing undesirable early activity of the polymerase at low temperatures that can lead to non specific amplification It allows users flexibility in reaction set up including the pre mixing of PCR reagents and storage at room temperature for up to 72 hours prior to cycling The polymerase is reactivated after only a 2 minute incubation at 95 C PowerUp SYBR Green Master Mix contains heat labile uracil DNA glycosylase UDG UDG is also known as uracil N glycosylase UNG Treatment with heat labile UDG can prevent the reamplification of carryover PCR products by removing any uracil incorporated into single or double stranded amplicons Longo et al 1990 Heat labile 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 11 for more information about UDG PCR products are stable for up to 72 hours post amplification using master mixes containing heat la
7. USER GUIDE applied biosystems PowerUp SYBR Green Master Mix Catalog Numbers A25741 A25742 A25743 A25776 A25777 A25778 A25779 A25780 A25918 Revision B 0 Publication Part Number MAN0013511 ThermoFisher For Research Use Only Not for use in diagnostic procedures SCIENTIFIC For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice DISCLAIMER TO THE EXTENT ALLOWED BY LAW LIFE TECHNOLOGIES AND OR ITS AFFILIATE S WILL NOT BE LIABLE FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT IMPORTANT LICENSING INFORMATION Limited Use Label License 477 Real Time PCR System Notice to Purchaser This product is licensed for use under certain patent claims owned by the University of Utah Research Foundation and licensed to BioFire Diagnostics Inc No right is conveyed expressly by implication or by estoppel under any other patent claim TRADEMARKS 2015 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified 2 PowerUp SYBR Green Master Mix User Guide Contents Product 4 Chemistry ao 6 Contents and Storage cecciciacicn cad cea tner ia la Sed dae ede dees A eee Seca ede eee 8 Required Materials 9 Prevent Contamination
8. a Cr value TM Use primers that contain dA nucleotides near the 37 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 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 Guidelines for Designing Primers on page 26 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 When preparing samples for PCR amplification e Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves e Change gloves whenever you suspect that they are contaminated e Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Po
9. ber Template fur Amplification controls non specific L mp sfication y E 4 NTC Target 5 a Amplification a 2 3 non specific 23 amplification Az 3 0 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 30 PowerUp SYBR Green Master Mix User Guide Safety Appendix B Chemical Safety To minimize the hazards of chemicals Guidelines e Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials e 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 SDS e 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 SDS e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS e Comply with all local state provincial or national laws and regu
10. bile UDG Unlike standard UDG heat labile UDG is completely inactivated prior to amplification A blend of dUTP dTTP is included to enable UDG activity and maintain optimal PCR results The SYBR Green dye detects PCR products by binding to double stranded DNA formed during PCR see Chemistry Overview section TM TM TM ROX Passive PowerUp SYBR Green Master Mix contains ROX dye Passive Reference 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 4 PowerUp SYBR Green Master Mix User Guide Real Time Instruments About This Protocol TM TM PowerUp SYBR Green Master Mix can be used to run experiments on the following Applied Biosystems Real Time PCR Systems e StepOne and StepOnePlus Real Time PCR Systems e 7500 and 7500 Fast Real Time PCR Systems e 7900HT and 7900111 Fast Real Time PCR Systems e ViiA 7 Real Time PCR System TM e QuantStudio family of Real Time PCR Systems This protocol provides e Background information about gene quantification assays e A list of equipment and materials for using the PowerUp SYBR Green Master Mix e Procedures for using the PowerUp SYBR Green Master Mix For details about specific procedures described in this protocol see Suppo
11. cation curve begins before the mi maximum baseline Decrease the End Cycle value oul 1 Start End Cycle 20 PowerUp SYBR Green Master Mix User Guide Threshold Settings Analyze the Results Relative Quantitation Method Resources for Data Analysis Threshold Set Correctly The threshold is set in the exponential phase of the amplification curve qa Threshold settings above or below the optimum increase the standard deviation of the replicate groups 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 Threshold Set Too High The threshold is set above the exponential Threshold 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 TM TM Using the PowerUp SYBR Green Master Mix you can perform two types of quantitation relative and absolute e Relative quantitation compares a target against an internal standard You can perform relative quantitation using either the standard curve method or the comparative Cr method e Absolute quantitation compares the Cr of an unknown sample aga
12. ctions Wear appropriate protective eyewear clothing and gloves PowerUp SYBR Green Mix User Guide 15 Prepare the PCR Reactions 1 Prepare the appropriate number of reactions according to the volumes in the following table Component 10 pL well 20 pL well PowerUp SYBR Green Master Mix 2X Forward and Reverse Primers Variable Variable cDNA template RNase free water Total Volume 10 uL 20 uL 5 yl 10 uL Variable Variable 1 For optimal performance in Fast and Standard modes use a range from 300 800 nM of each primer For optimal performance use up to 100 ng of cDNA for each reaction Mix the components thoroughly and centrifuge 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 Seal the plate with an optical adhesive cover and centrifuge the plate briefly to spin down the contents and eliminate any air bubbles Note PCR can be performed on the reaction plate at any time up to 24 hours after completing the reaction setup when kept at room temperature PowerUp SYBR Green Master Mix User Guide Run the PCR Reaction Plate Run the plate on an Applied Biosystems real time quantitative PCR instrument See the appropriate instrument user guide for help with programming the thermal cycling conditions or with running the plate 1 Place the reaction plate in the in
13. efer to the instrument user manual for calibration instructions Note It is recommended to calibrate your instrument every 6 months 3 Load the plate into a Applied Biosystems real time PCR system Program the thermal cycling conditions according to the information in step 2 on page 17 Run the plate Compile the results for AR and Cr then select the minimum forward and reverse primer concentrations that yield the maximum AR values and low Cr values Dissociation curves help you select the optimal primer concentrations for your TM SYBR quantification assays 1 Review the linear view of the amplification plot in your NTC wells Note In Figure A 1 on page 30 part a the strong amplification of the NTC wells indicates that significant nonspecific amplification is occurring 2 Generate a dissociation curve with your Real Time PCR System Note In the example dissociation curve data shown in Figure A 1 on page 30 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 is typical of primer dimer formation and it indicates that lower primer concentration may provide optimal results PowerUp SYBR Green Mix User Guide 29 Example of Nonspecific Amplification a Target Amplification a oF 4 NTC nonspecific amplification D 1 5 10 15 20 25 4 3 4 Cycle Num
14. er Mix you need to synthesize single stranded cDNA from total RNA or mRNA samples For optimal performance the RNA should be e Between 0 002 and 0 2 ug uL e Less than 0 005 of genomic DNA by weight TM e Free of inhibitors of reverse transcription and PCR e Dissolved in PCR compatible buffer e Free of RNase activity IMPORTANT If you suspect that the RNA contains RNase activity add RNase inhibitor to the reverse transcription reaction at a final concentration of 1 0 U pL e Nondenatured e IMPORTANT It is not necessary to denature the RNA Denaturation of the RNA may reduce the yield of cDNA for some gene targets Use both of the following methods to examine DNA quality e 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 Azs0 Azgo 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 Template quantitation is critical for successful PCR reactions The 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 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 wavelengt
15. exer MLS 10 PowerUp SYBR Green Master Mix User Guide Prevent Contamination and Nonspecific Amplification Overview Using UDG to Minimize Reamplification Carryover Products Using NTC Controls Design Primers to Avoid Primer Dimers PCR Good Laboratory Practices 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 PowerUp SYBR Green Master Mix contains heat labile uracil DNA glycosylase UDG UDG is also known as uracil N glycosylase UNG Treatment with heat labile UDG is useful in preventing the reamplification of carryover PCR products The heat labile UDG used in the PowerUp SYBR Green Master Mix is a 26 kDa recombinant enzyme derived from the thermolabile UDG gene isolated from marine bacteria and expressed in E coli 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 thereby creating an alkali sensitive apyrimidic site in the DNA The enzyme has no activity on RNA or dT containing DNA Longo et al 1990 No Template Control NTC reactions can be used to identify PCR contamination NTC reactions contain all reaction components PowerUp SYBR Green Master Mix primers water except sample and therefore should not return
16. gov e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e 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 32 PowerUp SYBR Green Master Mix User Guide Documentation and Support Support You can download the following documents from thermofisher com lifescience Documents Document Part number a 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 1376785 Relative Standard Curve and Comparative Cr AACr Experiments Applied Biosystems StepOne Real Time PCR System Getting Started Guide for 4376784 Standard Curve Experiments Applied Biosystems StepOne Real Time PCR System Installation Maintenance and 4376782 Networking Guide 7500 Fast System Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and 4347828 Maintenance Getting Started Guide Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Relative
17. h is assumed to be 260 nm unless stated otherwise A260 values can be converted into pg L using Beer s Law Absorbance 260 nm sum of extinction coefficient contributions x cuvette pathlength x concentration The following formulas are derived from Beer s Law Ausubel et al 1998 e Concentration of single stranded DNA Azo x 33 Hg pL e Concentration of double stranded DNA 4260 x 50 pg pL e Concentration of single stranded RNA A2 x 40 pg pL Note Absorbance measurements of highly concentrated O D gt 1 0 or very dilute O D lt 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 e Store purified RNA templates at 20 or 70 C in RNase free water e Store purified DNA templates at 20 C or 70 C in TE pH 8 0 PowerUp SYBR Green Master Mix User Guide Set Up the Plate Document Select a Plate for PCR Configure the Plate Document Select a plate appropriate for your real time instrument Refer to page 9 for part numbers of the plates For information about configuring plate documents when performing real time quantification refer to the appropriate user guides listed in Support Documents on page 33 Prepare the PCR Reaction Plate General Guidelines Reminder About Your Primers Reagent Handling and Preparation e For best results it is recommended to perform four re
18. ided by a fume hood Read the SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 2 Run the gel For PCR fragments lt 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 3 Runsamples 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 PowerUp SYBR Green Mix User Guide 23 Troubleshoot Observation Possible Cause Action High Cr values poor Insufficient cDNA template is Use up to 100 ng of cDNA precision or failed PCR present template per 20 uL reaction reactions Quality of cDNA template is poor gt Quantify the amount of cDNA template e Test the cDNA template for the presence of PCR inhibitors Sample degradation Prepare fresh cDNA then repeat the experiment Incorrect pipetting Reduced number of PCR cycles in the thermal cycler protocol Prepare the reactions as described on page 16 Increase the number of PCR cycles to the default setting of 40 see page 17 Primer dimer formation and residual polymerase activity e Prepare the reaction mixes and the reaction plate on ice e 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 reac
19. ilable at thermofisher com techresources support The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available at thermofisher com techresources support Life Technologies and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on the Life Technologies website at http www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies 34 PowerUp SYBR Green Master Mix User Guide For support visit thermofisher com techresources or email techsupport lifetech com thermofish est ThermoFisher ermotisner com utescience 5 CIENTIFIC 28 July 2015
20. inst a standard curve with known copy numbers 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 For more information about analyzing your data refer to the appropriate instrument manual available at thermofisher com lifescience or contact Technical Support see page 33 for a list of support documents PowerUp SYBR Green Mix User Guide 21 Detect Nonspecific Amplification Because SYBR Green dye detects any double stranded DNA check for nonspecific product formation by using dissociation curve or gel analysis Dissociation 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 Curves probe interacting with double stranded DNA is plotted against temperature When to Generate Dissociation Curves Note Because of the presence of heat labile UDG
21. 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 or SuperScript VILO cDNA Synthesis Kit see page 10 2 Inthe PCR step PCR products are synthesized from cDNA samples using the PowerUp SYBR Green Master Mix Extension of primer on MRNA 8 3 mRNA RT rr 5 cDNA Random Primer Step Oligo d T Primer or Synthesis of 1st cDNA strand Random Hexamer 3 oo 5 CDNA Extension of primer on cDNA F 5 Forward Primer v Synthesis of 2nd cDNA strand 5 5 PCR Step 5 m 3 PCR amplification of cDNA N 5 Forward Primer 3 5 v y 2 5 E eA 5 a SS 5 Figure 2 Two step RT PCR PowerUp SYBR Green Mix User Guide Contents and Storage TM TM Contents The PowerUp SYBR Green Master Mix is supplied in a 2X concentration Catalog no Contents A25741 PowerUp SYBR Green Master Mix 1 mL A25779 PowerUp SYBR Green Master Mix 2 x 1 mL A25780 PowerUp SYBR Green Master Mix 5 x 1 mL A25918 PowerUp SYBR Green Master Mix 10 x 1 mL A25742 PowerUp SYBR Green Master Mix 5 mL A25776 PowerUp SYBR Green Master Mix 2 x 5 mL A25777 PowerUp SYBR Green Master Mix 5 x 5 mL A25778 PowerUp SYBR Green Master Mix 10 x 5 mL A25743 PowerUp SYBR Green Master Mix 50 mL Storage Store the PowerUp
22. l amplification curve as shown below has a Plateau phase a Linear phase b Exponential geometric phase c Background d Baseline e a a w 5 5 5 a PowerUp SYBR Green Mix User Guide 19 Manually Adjust 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 the Baseline and Threshold Reviewing all baseline and threshold values after analysis of the study data is recommended 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 Baseline Settings See the example amplification plots below to determine whether or not the baseline and threshold settings were correctly set evel som Plat Baseline Set Correctly me The amplification curve begins after the maximum baseline No adjustment necessary Orie Start End Cycle Prot Baseline Set Too Low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value Amic Phat Baseline Set Too High The amplifi
23. lations related to chemical storage handling and disposal Chemical Waste To minimize the hazards of chemical waste Safety Guidelines Read and understand the Safety Data Sheets SDSs 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 For additional safety guidelines consult the SDS 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 SDS 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 PowerUp SYBR Green Mix User Guide 31 Waste Disposal Biological Hazard Safety If potentially hazardous waste is generated
24. m Seal StepOne system plates with MicroAmp 48 Well Optical Adhesive Film Item Catalog number MicroAmp 48 Well Optical Adhesive Film e 25 covers 4375928 e 100 covers 4375323 MicroAmp Optical Adhesive Film e 25 covers 4360954 e 100 covers 4311971 PowerUp SYBR Green Mix User Guide Other Kits Item Catalog number High Capacity cDNA Reverse Transcription Kit e 200 reactions 4368814 e 200 reactions with RNase Inhibitor 4374966 e 1000 reactions 4368813 e 1000 reactions with RNase Inhibitor 4374967 High Capacity RNA to cDNA Kit e 50 reactions 4387406 SuperScript VILO cDNA Synthesis Kit e 50 reactions 4453650 e 250 reactions 4453651 Other IMPORTANT Do not use plastics made of polyethylene terephthalate co Consumables polyester glycol modified PTEG for storage of PowerUp SYBR Green Master Mix or reaction mixes SYBR dye has been shown to stick to this type of plastic material Plastics recommended for storage include polypropylene high density polyethylene HDPE and polystyrene Item Source Centrifuge with adapter for 96 well plates Major laboratory supplier MLS or Centrifuge with adapter for 384 well plates Disposable gloves MLS Microcentrifuge MLS Pipette tips with filter plugs MLS Pipettors positive displacement or air displacement MLS Polypropylene tubes MLS Tris EDTA TE Buffer pH 8 0 MLS Vort
25. n two G and or C bases If the template is Then DNA plasmid DNA genomic DNA cDNA Design the primers as described above Design the primers as described above Also see Select an Amplicon Site for cDNA on page 27 RNA Design the primers as described above 26 PowerUp SYBR Green Master Mix User Guide Select an Selecting a good amplicon site ensures amplification of the target cDNA Amplicon Site for without co amplifying the genomic sequence pseudogenes and related genes cDNA 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 Cr with cDNA and no amplification with no template control or genomic DNA If no 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 PowerUp SYBR Green Mix User Guide 27 Optimize Primer Concentrations for PCR Overvie
26. plicates of each reaction e Reaction mixes can be prepared depending upon your experimental requirements Scale the components to be included in the reaction mix according to the number of reactions to be performed Include an additional 10 of the reaction mix volume to account for variations in pipetting e If using smaller reaction volumes scale all components of the reaction mix proportionally Reaction volumes lt 10 uL are not recommended Refer to page 26 for information about identifying target sequences and designing primers Note Separate PCR thermal cycling conditions are required for primers with a Tm lt 60 C Follow these guidelines to ensure optimal PCR performance Prior to use TM TM e Mix the PowerUp SYBR Green Master Mix thoroughly by swirling the bottle e Place frozen cDNA samples and primers on ice to thaw After the samples are thawed vortex them then centrifuge the tubes briefly e IMPORTANT Do not use plastics made of polyethylene terephthalate co polyester glycol modified PTEG for storage of PowerUp SYBR Green Master Mix or reaction mixes SYBR dye has been shown to stick to this type of plastic material Plastics recommended for storage include polypropylene high density polyethylene HDPE and polystyrene CHEMICAL HAZARD PowerUp SYBR Green Master Mix 2X may cause eye skin and respiratory tract irritation Read the SDS and follow the handling instru
27. r up to 24 hours later if the plate is stored exposed to light 4 Set the reaction volume appropriate for the type of plate being used for your PCR reaction 5 Start the run PowerUp SYBR Green Mix User Guide 17 Analyze Your Results The general process for analyzing the data from gene expression assays requires that you e View the amplification plots e Adjust the baseline and threshold values to determine the threshold cycles Cr for the amplification curves e Use the standard curve method or the relative quantification AACr method to analyze the results Use the software provided with your instrument to automatically calculate or Baseline and manually set the baseline and threshold for the amplification curves Threshold Values e Baseline refers to the initial cycles of PCR in which there is little change in fluorescence signal e The intersection of the threshold with the amplification plot defines the Cr in real time PCR assays The threshold is set above the background and within the exponential growth phase of the amplification curve Rn Threshold Baseline Cy T T T 0 5 10 15 20 25 30 35 40 Cycle Number 18 PowerUp SYBR Green Master Mix User Guide View the The instrument software calculates baseline and threshold values for a detector Amplification based on the assumption that the data exhibit the typical amplification curve Plots A typica
28. rt Documents on page 33 PowerUp SYBR Green Mix User Guide Chemistry Overview How the SYBR Green Dye Chemistry Works The SYBR Green dye is used to detect PCR products by binding to double stranded DNA formed during PCR The process works as follows 1 When PowerUp SYBR Green Master Mix is added to a sample SYBR Green dye immediately binds to all double stranded DNA TM 2 During the PCR Dual Lock DNA Polymerase amplifies the target sequence which creates the PCR product or amplicon 3 The SYBR Green dye then binds to each new copy of double stranded DNA As the PCR progresses more amplicon is created Because the SYBR Green 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 following figure illustrates this process Step 1 The SYBR Green dye During PCR Dual The SYBR Green dye within the PowerUp Lock DNA Polymerase then binds to each new SYBR Green Master amplifies each target copy of double Mix immediately binds stranded DNA with all double stranded DNA Figure 1 Representation of how the SYBR Green dye acts on double stranded DNA during one extension phase of PCR PowerUp SYBR Green Master Mix User Guide Using the Master When performing a two step RT PCR reaction total or mRNA must first be Mix in Two Step transcribed
29. strument 2 Set the thermal cycling conditions using the default PCR thermal cycling conditions specified in the following tables according to the instrument cycling parameters and the melting temperature of your primers Fast Cycling Mode Primer Tm 260 C Step Temperature Duration Cycles UDG Activation 50 C 2 min Hold tea DNA 95 C Jin Hold Denature 95 C 1 or 3 sec 40 Anneal Extend 60 C 30 sec Denature for 1 second when using ViiA 7 or QuantStudio family of Real Time PCR Systems Denature for 3 seconds when using 7500 Fast StepOne or StepOnePlus Real Time PCR Systems Standard Cycling Mode Primer Tm 260 C Step Temperature Duration Cycles UDG Activation 50 C 2 min Hold Drak Loe DNA 95 C 2 min Hold Polymerase Denature 95 C 15 sec 40 Anneal Extend 60 C 1 min Standard Cycling Mode Primer Tm lt 60 C Step Temperature Duration Cycles UDG Activation 50 C 2 min Hold aes bas 95 C 2 min Hold Denature 95 C 15 sec Anneal 55 60 C 15 sec 40 Extend 72 C 1 min For best results only use the standard cycling mode with 7900HT Real Time PCR Instruments Anneal temperature should be set to the melting point for your primers 3 Set the instrument to perform a default dissociation step Note A dissociation curve can be performed up to 72 hours after the real time PCR run if the plate is stored in the dark o
30. sure that the reaction plate is sealed completely especially around the edges High variability across Reaction mix was not mixed well Mix the reaction mix gently by replicates inversion then centrifuge briefly before aliquoting to the reaction plate Fluorescent intensity too high Primer concentration is too high Use lt 200 nM of each primer StepOne and StepOnePlus systems PowerUp SYBR Green Mix User Guide 25 Appendix A Identify Target Sequences and Design Primers Identify Target A target template is a DNA sequence including cDNA genomic DNA or Sequence and plasmid nucleotide sequence that you want to amplify Amplicon Size 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 Guidelines for Using Primer Express Software Designing Primers 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 e Donot overlap primer and probe sequences The optimal primer length is 20 bases e Keep the GC content in the 30 70 range e Avoid runs of identical nucleotides If repeats are present there must be fewer than four consecutive G residues e Make sure the last five nucleotides at the 3 end contain no more tha
31. tion plate at 4 C Low AR or R values Extension time is too short Use the default thermal profile settings see page 17 Primer dimer formation and residual polymerase activity e Prepare the reaction mixes and the reaction plate on ice e 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 24 PowerUp SYBR Green Master Mix User Guide Observation Possible Cause Action R vs Cycle plot is not ROX dye was not selected as the Select ROX dye as the passive displayed passive reference when the plate reference when you set up the document was set up plate document Extremely high AR or Rn ROX dye was not selected as the Select ROX dye as the passive values passive reference when the plate reference when you set up the document was set up 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 Select ROX dye as the passive passive reference when the plate reference when you set up the document was set up plate document Evaporation Make
32. w By independently varying the forward and reverse primer concentrations you can identify the primer concentrations that provide optimal assay performance The primer concentrations you select should provide a low Cr and a high AR when run against the target template but should not produce nonspecific product formation with NTCs Quantitate the 1 Measure the absorbance at 260 nm of a 1 100 dilution of each primer Primers oligonucleotide in TE buffer 2 Calculate the sum of extinction coefficient contributions for each primer extinction coefficient contribution X extinction coefficient x number of bases in oligonucleotide sequence See An Example Calculation of Primer Concentration on page 28 for an example calculation 3 Calculate the oligonucleotide concentration in pM for each primer absorbance at 260 nm sum of extinction coefficient contribution x cuvette pathlength x concentration 100 Rearrange to solve for concentration concentration 100 absorbance at 260 nm sum of extinction coefficient contribution x cuvette pathlength 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 Chromophore Extinction Number of Specific Extinction Coefficient Chromophores in Example Coefficient Sequence Contribution A 15 200 1 15 200 0 7050 6 42 300
33. werUp SYBR Green Mix User Guide 11 PCR Good e Never bring amplified PCR products into the PCR setup area Laboratory Practices Continued e Open and close all sample tubes carefully Try not to splash or spray PCR samples e Keep reactions and components capped as much as possible gt Use a positive displacement pipette or aerosol resistant pipette tips e Clean lab benches and equipment periodically with a 10 bleach solution 12 PowerUp SYBR Green Master Mix User Guide Methods Procedural Overview This diagram is an overview of the procedures for performing gene expression experiments Prepare total RNA or Ti mi Perform reverse transcription Real Time PCR instrument Thermal cycler E Create and set up a plate document M gt SDS software 2 8 oo a Prepare the PCR reaction plate oF or 5 i 384 well plate 96 well 48 well Plate o Standard Fast plate a Run the PCR reaction plate Real Time PCR ir instrument Analyze results Amplification Plot PowerUp SYBR Green Mix User Guide Prepare the Template Examine RNA Template Quality Examine DNA Template Quality Quantitate the Template Store the Template After isolating the template examine its quality and quantity and store it properly Before using the PowerUp SYBR Green Mast
34. 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 e Ensure the health and safety of all personnel in your laboratory e 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 AN MAINE 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 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 e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories http bmbl od nih
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