SYBR® Select Master Mix for CFX User Guide (PN 4474514A)
Contents
1. 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 C 7050 6 42 300 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 Mcm x 0 3 cm x C 100 C 258 uM 28 SYBR Select Master Mix for CFX User Guide Determine the Optimal Primer Concentration Confirm the Absence of Nonspecific Amplification AN Nailed CHEMICAL HAZARD SYBR Select Master Mix for CFX 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 SYBR Select Master Mix for CFX is 1X Note The plate conf2. guidelines published in Biosafety in Microbiological and Biomedical Laboratories http bmbl od nih 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 SYBR Select Master Mix for CFX User Guide Documentation and Support Support You can download the following documents from the Life Technologies website Documents at www lifetechnologies com Document Part number High Capacity CDNA Reverse Transcription Kit Protocol 4375575 Primer Express Software Version 3 0 Getting Started Guide 4362460 Obtaining support For the latest services and support information for all locations go to www lifetechnologies com At the website you can Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support techsupport lifetech com Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and pat
3. variation is typical of primer dimer formation and it indicates that lower primer concentration may provide optimal results SYBR Select Master Mix for CFX User Guide 29 Example of Nonspecific Amplification Amplification Target Amplification Figure A 1 Amplification data using SYBR Green dye chemistry a Amplification plot linear view demonstrating suspected nonspecific amplification in NTC wells b Melt curve analysis confirming that product in NTC wells has a melting temperature different from the specific product 30 SYBR Select Master Mix for CFX 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 See About SDSs on page Error Bookmark not defined 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 procedure
4. USER GUIDE applied biosystems oy Lie technologies SYBR Select Master Mix for CFX Catalog Numbers 4472937 4472942 4472952 4472953 4472954 and 4472947 Revision Date 23 April 2012 Rev A Publication Part Number 4474514 For Research Use Only Not intended for any animal or human therapeutic or diagnostic use technologies For Research Use Only Not intended for animal or human therapeutic or diagnostic use Information in this document is subject to change without notice LIFE TECHNOLOGIES DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT LIFE TECHNOLOGIES IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PUCHASER LIMITED LICENSE End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand alone product or as a component of another product LIMITED USE LABEL LICENSE RESEARCH USE ONLY The purchase of this product conveys to the purchaser the l
5. 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 SYBR Select Master Mix for CFX User Guide PCR Good e Never bring amplified PCR products into the PCR setup area Laboratory e Open and close all sample tubes carefully Try not to splash or spray PCR Practices samples Continued e Keep reactions and components capped as much as possible e Use a positive displacement pipette or aerosol resistant pipette tips e Clean lab benches and equipment periodically with a 10 bleach solution SYBR Select Master Mix for CFX User Guide Methods Procedural Overview This diagram is an overview of the procedures for performing gene expression experiments Prepare total RNA Perform reverse transcription Create and set up a plate document Prepare the PCR reaction plate 96 well plate Run the PCR reaction plate Real Time PCR instrument Thermal cycler or PCR Amplification HE Z Analyze results Su Va Amplification Pl
6. bitor 4374966 e 1000 reactions 4368813 e 1000 reactions with RNase Inhibitor 4374967 SuperScript VILO cDNA Synthesis Kit e 50 reactions 4453650 e 250 reactions 4453651 Other Consumables 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 Vortexer MLS SYBR Select Master Mix for CFX 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 SYBR Select Master Mix for CFX 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 SYBR Select Master Mix for CFX 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
7. c 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 wavelength is assumed to be 260 nm unless stated otherwise A260 values can be converted into pg pL 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 Azs x 33 ug uL e Concentration of double stranded DNA Azs x 50 ug uL e Concentration of single stranded RNA Azs x 40 ug 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 SYBR Select Master Mix for CFX User Guide 13 Store the Store the templates as follows Template e Store purified RNA templates at 20 C or 70 C in RNase free water e Store purified DNA templates at 20 C or 70 C in TE pH 8 0 14 SYBR Select Master Mix for CFX User Guide Set Up the Pla
8. check the migration of the samples SYBR Select Master Mix for CFX User Guide 23 Troubleshoot Observation Possible Cause Action High Cy 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 e 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 Prepare the reactions as described on page 16 Reduced number of PCR cycles in Increase the number of PCR the thermal cycler protocol cycles to the default setting of 40 see page 17 Primer dimer formation and e Prepare the reaction mixes residual polymerase activity 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 Low RFU 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
9. ches Safety Data Sheets Safety Data Sheets SDSs are available at www lifetechnologies com support SDS Certificate of The Certificate of Analysis provides detailed quality control and product Analysis qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box SYBR Select Master Mix for CFX User Guide 33 Headquarters 9 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www appiredbkiuspsteappom suppwuril techsupport invitrogen com technologies www lifetechnologies com
10. 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 SYBR Select Master Mix for CFX primers water except sample and therefore should not return a Cr value 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 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
11. gs 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 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 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 RFU Cycles Baseline Set Too Low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value Amplification Baseline Set Too High The amplification curve begins before the maximum baseline Decrease the End Cycle value Amplification SE RFU SAA 20 SYBR Select Master Mix for CFX User Guide Threshold Settings Analyzing the Results Relative Quantitation Method Resources for Data Ana
12. hours after completing the reaction setup store the reaction plate at 4 C 24 SYBR Select Master Mix for CFX User Guide Observation Possible Cause Action Extremely high RFU values Evaporation Make sure that the reaction plate is sealed completely especially around the edges Lower RFU values obtained in early cycles C value is less than 15 Adjust the upper baseline range to a value less than 15 High variability across the reaction plate 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 SYBR Select Master Mix for CFX 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 Get
13. iguration 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 150 200 400 150 150 150 200 150 400 150 200 150 200 200 200 400 200 400 150 400 200 400 400 400 2 Calibrate your instrument for SYBR Green Dye if necessary Refer to the instrument user manual for calibration instructions Note It is recommended to calibrate your instrument every 6 months 3 Load the plate into the CFX real time PCR detection system Program the thermal cycling conditions according to the information in step 2 on page 17 Run the plate Compile the results for RFU and C then select the minimum forward and reverse primer concentrations that yield the maximum RFU values and low C values Melt curves help you select the optimal primer concentrations for your 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 melt curve with your Real Time PCR System Note In the example melt 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
14. imited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Human diagnostic uses require a separate license from Roche The right to human in vitro diagnostics requires a separate license For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners AmpliTaq is a registered trademark of Roche Molecular Systems Inc CFX96 Touch and CFX384 Touch are trademarks of Bio Rad Laboratories Inc O 2012 Life Technologies Corporation All rights reserved 2 SYBR Select Master Mix for CFX User Guide Contents Product info mirta AA AAA AAA anda 4 Chemistry ro 6 Contents and Sto
15. lysis Threshold Set Correctly Ampittcaton 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 w Lado RFU Threshold Set Too Low Amplification The threshold is set below the exponential phase of the amplification curve The w standard deviation is significantly higher than that for a plot where the threshold is w set correctly Set the threshold up into the exponential phase of the curve 10 Hades RFU Threshold Set Too High Amplification 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 Using the SYBR Select Master Mix for CFX 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 Cy method e Absolute quantitation compares the Cy of an unknown sample against 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
16. n ice to thaw After the samples are thawed vortex them then centrifuge the tubes briefly N OZVI CHEMICAL HAZARD SYBR Select Master Mix for CFX 2X may cause eye skin and respiratory tract irritation Read the SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 1 Prepare the appropriate number of reactions according to the volumes in the following table Component 384 Well Plate 96 Well Plates 10 pL well 20 uL well SYBR Select Master Mix for CFX 2X apl UL Forward and Reverse Primers Variable Variable EDNA template RNase free Variable Variable water Total Volume 10 pL 20 pL t For optimal performance use a range from 150 400 nM of each primer For optimal performance use up to 100 ng of cDNA for each reaction 2 Mix the components thoroughly and centrifuge briefly to spin down the contents and eliminate any air bubbles 3 Transfer the appropriate volume of each reaction to each well of an optical plate 4 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 72 hours after completing the reaction setup when kept at room temperature SYBR Select Master Mix for CFX User Guide Run the PCR Reaction Plate Run the plate with the CFX real time PCR system See the appr
17. o all double stranded DNA 2 During the PCR AmpliTaq DNA Polymerase UP amplifies the target sequence which creates the PCR product or amplicon 3 The SYBR GreenER dye then binds to each new copy of double stranded DNA 4 As the PCR progresses more amplicon is created Because the SYBR GreenER 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 GreenER During PCR AmpliTaq The SYBR GreenER dye within the SYBR DNA Polymerase UP dye then binds to each Select Master Mix for amplifies each target new copy of double CFX immediately binds stranded DNA with all double stranded DNA Figure 1 Representation of how the SYBR GreenER dye acts on double stranded DNA during one extension phase of PCR SYBR Select Master Mix for CFX User Guide Using the Master When performing a two step RT PCR reaction total or mRNA must first be Mix in Two Step transcribed into cDNA RT PCR 1 Inthe 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 9 2 Inthe PCR step PCR products are synthesized from cDNA samples using the SYBR Select Master Mix for CFX Extension of p
18. on 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 AAC 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 Cy in real time PCR assays The threshold is set above the background and within the exponential growth phase of the amplification curve RFU Threshold Baseline Cy T T T 0 5 10 15 20 25 30 35 40 Cycle Number 18 SYBR Select Master Mix for CFX 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 typical amplification curve as shown below has a e Plateau phase a e Linear phase b e Exponential geometric phase c e Background d e Baseline e Amplification 10 Threshold SYBR Select Master Mix for CFX User Guide 19 Manually Adjust the Baseline and Threshold Baseline Settin
19. opriate 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 tables according to the melting temperature of your primers Standard Cycling Mode Primer T 260 C Step Temperature Duration Cycles UDG Activation 50 C 2 min Hold AmpliTaq DNA Polymerase UP 95 C 2 min Hold Activation Denature 95 C 15 sec 40 Anneal Extend 60 C 1 min Standard Cycling Mode Primer T lt 60 C Step Temperature Duration Cycles UDG Activation 50 C 2 min Hold AmpliTaq DNA Polymerase UP 95 C 2 min Hold Activation Denature 95 C 15 sec Anneal 55 60 C 15 sec 40 Extend 72 C 1 min Anneal temperature should be set to the melting point for your primers 3 Set the instrument to perform a default dissociation step Note A melt curve can be performed up to 72 hours after the real time PCR run if the plate is stored in the dark or 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 SYBR Select Master Mix for CFX User Guide 17 Analyze Your Results The general process for analyzing the data from gene expressi
20. ot 12 SYBR Select Master Mix for CFX User Guide Prepare the Template Examine RNA Template Quality Examine DNA Template Quality Quantitate the Template After isolating the template examine its quality and quantity and store it properly Before using the SYBR Select Master Mix for CFX 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 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 Adding RNase inhibitor to the reverse transcription reaction is not necessary if the RNA is purified using the 6100 Nucleic Acid PrepStation and nucleic acid purification reagents 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 e Spectrophotometry The Axo Az9 ratio should be 1 8 to 2 0 Smaller ratios usually indicate contamination by protein or organi
21. r Guide 31 Waste Disposal Biological Hazard Safety If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory 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 WAS 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
22. r dimers are most prevalent in NTC wells and sample wells containing a low concentration of template Melt Peak Specific Product pisos ke Primer Dimer d RFU dT Temperature Celsius Figure 3 Example of two melt curves 22 SYBR Select Master Mix for CFX 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 provided 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 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
23. rage runs sist cto tna ia da as A eee ig hala 8 Required Material niiina iea ae ae daa badet 9 Prevent Contamination and Nonspecific Amplification ssrrrnrrnrnnrrrrnvnnrnrrnrnnnrnnnnrnnnnrnnnnrrnnnnrnnnernnnnnnnnn 10 Methods a E T A E ee keddadanded 12 Procedural Overview Nna aR E EEE E ETEN 12 Prepare the Template seccion cios ennienni ener ekee lapa det stadnamn kokken toi 13 Set Up the Plate Documento ccoo dad dannes 15 Pr pare the PCR Reaction Plate ocio suse irte cast eee teeta e Da are et ins 16 Run the PCR Reaction Plate ati 17 Analyze Your ResultSi s snertne ap dead been ed pek dnne dene aten eee 18 Detect Nonspecific Amplification rnrnnnnnrnnrnrnnrnrnnrnrrnnnrrnnnnrnnnnrn nen renn norr aner aner nnnn renn nnnrnnnnrnnnernnnnnnnnn 22 Trouble hot la we A Aa Ae E ad Aa Ae ee a 24 A AO 26 Identify Target Sequences and Design Primers ccc ccceecceeeeeeeceeeceeeeeeseceeseaeeeeaeeecaeeeseeeseeeeseeeeeneeeeaes 26 Optimize Primer Concentrations for PCR rnrrrnnnnrnrnnrnnrnrnnrnrnnnnnrnnnnrnnnernnnnrnnnnnrn venns noen nnernnnernnnnrrnnnnrnnnernnn 28 Appendix BB xiii A i n 31 SA RR a a A AS A A da a a I 31 Documentationand Sapport aassren dann rene ideene utdeles net 33 SYBR Select Master Mix for CFX User Guide 3 Product Information About the Reagent Hot Start UDG dUTP dTTP SYBR GreenER The SYBR Select Master Mix for CFX is formulated to provide superior specificity and sensitivi
24. 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 from the instrument manufacturer SYBR Select Master Mix for CFX User Guide 21 Detect Nonspecific Amplification Melt Curves Because SYBR GreenER dye detects any double stranded DNA check for nonspecific product formation by using melt curve or gel analysis A melt curve is a graph that displays melt 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 Melt Curves Note Because of the presence of heat labile UDG you can generate a melt curve up to 72 hours after the real time PCR run An Example The melt 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 Prime
25. rimer on MRNA a 3 mRNA RT SS cDNA Random Primer Step Oligo d T Primer or Synthesis of 1st cDNA strand Random Hexamer 3 5 DNA Extension of primer on cDNA 3 5 Forward e Primer o Synthesis of 2nd cDNA strand amp 5 PCR Step 5 m 3d PCR amplification of cDNA S Forward Primer EN 3 5 2 S 5 a Reverse a Primer 5 Figure 2 Two step RT PCR SYBR Select Master Mix for CFX User Guide Contents and Storage Contents The SYBR Select Master Mix for CFX is supplied in a 2X concentration Item Part Number Contents Mini Pack 4472937 One 1 mL tube 100 x 20 uL reactions 1 Pack 4472942 One 5 mL tube 500 x 20 uL reactions 2 Pack 4472952 2 x 5 mL tubes 1000 x 20 uL reactions 5 Pack 4472953 5 x 5 mL tubes 1500 x 20 uL reactions 10 Pack 4472954 10 x 5 mL tubes 5000 x 20 uL reactions Bulk Pack 4472947 One 50 mL tube 5000 x 20 uL reactions Storage Store the SYBR Select Master Mix for CFX at 2 C to 8 C SYBR Select Master Mix for CFX User Guide Required Materials Plates and Optical Refer to the manual supplied by the instrument manufacturer for details on Seals selecting the plate appropriate for your real time instrument Seal plates with the appropriate optical adhesive film Other Kits Item Catalog number High Capacity cDNA Reverse Transcription Kit e 200 reactions 4368814 e 200 reactions with RNase Inhi
26. s as recommended in the SDS e Comply with all local state provincial or national laws and regulations 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 SYBR Select Master Mix for CFX Use
27. 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 SYBR Select Master Mix for CFX User Guide 27 Optimize Primer Concentrations for PCR Overview Quantitate the Primers 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 C and a high RFU 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 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
28. te Document Select a Plate for Refer to the manual supplied by the instrument manufacturer for details on PCR selecting the plate appropriate for your real time instrument Configure the For information about configuring plate documents when performing real time Plate Document quantification refer to the appropriate user guide supplied by the manufacturer SYBR Select Master Mix for CFX User Guide 15 Prepare the PCR Reaction Plate General Guidelines Reminder About Your Primers Reagent Handling and Preparation Prepare the PCR Reactions e For best results it is recommended to perform four replicates 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 e Mix the SYBR Select Master Mix for CFX thoroughly by swirling the bottle e Place frozen cDNA samples and primers o
29. ter mixes containing heat labile 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 GreenER dye detects PCR products by binding to double stranded DNA formed during PCR see Chemistry Overview section The SYBR GreenER dye provides both higher sensitivity and lower PCR inhibition than SYBR Green I dye It can be used on real time PCR instruments calibrated for SYBR Green I dye without any change of filters or settings SYBR Select Master Mix for CFX User Guide Real Time SYBR Select Master Mix for CFX can be used to run experiments with the CFX Instruments Real Time PCR Detection Systems e CFX96 Touch Real Time PCR Detection System e CFX384 Touch Real Time PCR Detection System About This This protocol provides Protocol e Background information about gene quantification assays e A list of equipment and materials for using the SYBR Select Master Mix for CFX e Procedures for using the SYBR Select Master Mix for CFX SYBR Select Master Mix for CFX User Guide Chemistry Overview How the SYBR The SYBR GreenER dye is used to detect PCR products by binding to double GreenER Dye stranded DNA formed during PCR The process works as follows Chemistry Works 1 When SYBR Select Master Mix for CFX is added to a sample SYBR GreenER dye immediately binds t
30. ting 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 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 above Also see Select an Amplicon Site for cDNA on page 27 RNA Design the primers as described above 26 SYBR Select Master Mix for CFX 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 C with cDNA and no amplification with no template control or genomic DNA If no good sequence is found you may need to examine the
31. ty It is supplied in a convenient 2X concentration premix to perform real time PCR using SYBR GreenER dye The master mix contains e SYBR GreenER Dye e AmpliTaq DNA Polymerase UP Ultra Pure with a proprietary hot start mechanism e Heat labile Uracil DNA Glycosylase UDG e dNTP blend containing dUTP dTTP e Optimized buffer components The user only needs to provide primers template and water The AmpliTaq DNA Polymerase UP is provided in an inactive state to automate the hot start PCR technique and allow flexibility in the reaction setup including pre mixing of PCR reagents at room temperature The polymerase is equipped with a proprietary hot start mechanism that provides improved specificity The polymerase is re activated after a 2 minute incubation at 95 C SYBR Select Master Mix for CFX 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 10 for more information about UDG PCR products are stable for up to 72 hours post amplification using mas
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