SYBR® Green PCR and RT-PCR Reagents
Contents
1. 50 300 300 300 900 300 900 50 900 300 900 900 900 Load the plate for both a template and a No Template Control NTC matrix as shown in Plate Configuration for Primer Optimization on page 14 Place the plate in the ABI PRISM 7700 Sequence Detector Use the thermal cycling conditions in Thermal Cycling Parameters for Primer Optimization on page 14 Confirm that a SYBR Green Pure Dye spectrum is accessed by the SDS software before starting the run At the end of the run Tabulate the results for the yield This analysis will identify the optimum concentrations of primers for PCR yield Tabulate the results for the Cy value This analysis will identify the optimum primer concentrations for Cy and for the absence of non specific amplification Note The absence of non specific amplification should be confirmed by analyzing the PCR products by agarose gel electrophoresis see page 9 15 Optimize Primer One step RT PCR reactions with MultiScribe Reverse Transcriptase are Concentrations for optimized following a similar protocol In this case the PCR Master Mix One Step RT PCR for Primer Optimization on page 13 is replaced with the corresponding One Step RT PCR Master Mix See the table below Note Procedures for one step RT PCR are the same as for two step RT PCR See Optimize Primer Concentrations for Two Step RT PCR on page 13 One Step RT PCR Maste2. Cycle Number eo Materials and Equipment Kit Contents The SYBR Green PCR Core Reagents P N 4304886 contain enough PCR reagents for up to 200 50 uL reactions Table 1 SYBR Green PCR Core Reagents Reagent Volume Description 10X SYBR Green 1 5 mL One tube containing optimized 10X SYBR Green PCR Buffer PCR Buffer which includes Passive Reference 1 AmpErase UNG 100 uL One vial containing 1 U uL uracil N glycosylase AmpliTaq Gold 50 uL One vial containing 5 U uL AmpliTaq Gold DNA Polymerase DNA Polymerase dNTP Mix 1 0 mL One tube containing 2 5 mM dATP 2 5 mM dCTP 2 5 mM dGTP and 5 0 mM dUTP 25 mM MgClo 3 0 mL Two tubes containing 25 mM MgClo 1 5 mL each Storage and Stability Store the SYBR Green PCR Core Reagents at 15 to 25 C If stored under the recommended conditions the product will maintain performance through the control date printed on the label MSDS For extra copies of Material Safety Data Sheets MSDS contact Customer Service at 1 800 345 5224 Materials Required But Not Supplied The following items are required in addition to the reagents supplied in the SYBR Green PCR Core Reagents Table 2 User supplied Materials Item Source ABI PRISM 7700 Sequence Detection System See your local PE Applied Biosystems representative for the instrument or software best suited to meet your needs Primer Express software single use license PE App
3. Action 3 Add the enzymatic components e g MultiScribe Reverse Transcriptase RNase Inhibitor and the RNA Mix the components by inverting the microcentrifuge tube Transfer the contents to a MicroAmp Optical tube or multiple wells of a MicroAmp Optical 96 Well Reaction Plate Using MicroAmp Optical Caps cap the tubes and briefly centrifuge to remove air bubbles and collect the liquid at the bottom of the tube Transfer the plates to the thermal cycler block Perform RT as described in Thermal Cycling Parameters for RT Reactions on page 12 Remove the 96 Well Reaction Plate after thermal cycling is complete Optimize Primer The purpose of this procedure is to determine the minimum primer Concentrations for concentrations giving the maximum AR and minimum non specific Two Step RT PCR amplification The reaction volumes are 50 uL Use 10 100 ng of genomic DNA or 1 10 ng of cDNA template The ABI PRISM 7700 Sequence Detector can provide additional data for optimization using the minimum threshold cycle C7 See Real Time Detection on page 3 for more information regarding Cr PCR Master Mix for Primer Optimization Volume per Volume per 100 50 pL Reactions Component Reaction pL uL 10X SYBR PCR Buffer 5 0 500 25 mM MgCl 6 0 600 dNTP Blend 2 5 mM dATP 2 5 mM 4 0 400 dCTP 2 5 mM dGTP 5 0 mM dUTP AmpliTaq Gold 5 U uL 0 25 25 AmpErase U
4. Organizing a laboratory for PCR work In Innis M A Gelfand D H Sninsky J J and White T J eds PCR Protocols A Guide to Methods and Applications Academic Press Inc San Diego CA pp 447 454 PCR Technical Information 1996 In Perkin Elmer Systems Reagents amp Consumables 1996 1997 Foster City CA Saiki R K Scharf S J Faloona F A Mullis K B Horn G T Erlich H A and Amheim N 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Saiki R K Gelfand D H Stoffel S Scharf S J Higuchi R Horn G T Mullis K B and Erlich H A 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 239 487 491 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Press 21 PE Applied Biosystems A DIVISION OF PERKIN ELMER THE AMERICAS United States PE Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 Tel 650 570 6667 800 345 5224 Fax 650 572 2743 Canada Mississauga Ontario Tel 905 821 8183 800 668 6913 Fax 905 821 8246 Latin America Del A Obregon Mexico Tel 52 5 651 7077 Fax 52 5 593 6223 EUROPE AFRICA Austria Wien Tel 01 602 3101 Fax 01 6025177 Benelux Nieuwerkerk a d IJssel Netherlands Tel 31 0 180 331400 Fax
5. amplicon size ranges from 50 150 bp Design primers using Primer Express software as described in the DNA PCR section of the Primer Express Applications Based Primer Design Software User s Manual P N 4303014 Follow these guidelines Primers can be designed as close as possible to each other provided that they do not overlap Keep the GC content in the 20 80 range Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more Gs should be avoided When using Primer Express software the Tm should be 58 60 C The five nucleotides at the 3 end should have no more than two G and or C bases Order Reagents See Materials Required But Not Supplied on page 4 for a list of required reagents and equipment Quantitate Use a spectrophotometric method to determine the concentrations of Primers the primers received Measure the absorbance at 260 nm of a 1 100 dilution of each oligonucleotide in TE buffer Calculate the oligonucleotide concentration C in uM using the method shown in Table 3 Table 3 Extinction Coefficient Calculation Extinction Extinction Coefficient Chromophore Coefficient Number Contribution A 15 200 1 15 200 C 7 050 6 42 300 G 12 010 5 60 050 T 8 400 6 50 400 Total 167 950 Absorbance 260 nm sum of extinction coefficient contributions x cuvette pathlength x oligonucleotide concentration 100 0 13 167 950 M c
6. emission intensity of SYBR Green by the emission intensity of the Passive Reference to obtain a ratio defined as the R normalized reporter for a given reaction R R value of a reaction containing all components including the template Ra Rn value of an unreacted sample This value can be obtained from the early cycles of a Real Time run those cycles prior to a detectable increase in fluorescence R can also be obtained from a reaction not containing template AR Rnt Rr where Emission Intensity of SYBR Green Rnt PCR with template Emission Intensity of Passive Reference Emission Intensity of SYBR Green PCR without template or early cycles of a Real Time reaction Emission Intensity of Passive Reference AR indicates the magnitude of the signal generated by the given set of PCR conditions Real Time Detection The threshold cycle or C4 value is the cycle at which a statistically significant increase in AR is first detected Threshold is defined as the average standard deviation of R for the early cycles multiplied by an adjustable factor On the graph shown below the threshold cycle occurs when the Sequence Detection Application begins to detect the increase in signal associated with an exponential growth of PCR product Threshold ht at eth aE No Template Control C Normalized Fluorescence Intensity GR1051 eo a a eo 15 20 25 30 35 4
7. 31 0 180 331409 Chekia Rep Praha Tel 2 61 22 21 64 Fax 2 61 22 21 68 Denmark Aller d Tel 48 100 400 Fax 48 100 401 Finland Espoo Tel 0880 144 Fax 0 803 8002 France Paris Tel 016959 85 85 Fax 01 69 59 85 00 P N 4304965 Rev A Germany Weiterstadt Tel 0 6150 101 0 Fax 0 6150 101 101 Hungary Budapest Tel 1 251 11 16 Fax 1 251 14 61 Italy Milano Tel 039 23831 Fax 039 2383490 Norway Oslo Tel 0 22 02 1500 Fax 0 22 02 1501 Poland Warszawa Tel 22 33 09 36 Fax 22 33 09 96 Russia Moskva Tel 095 935 8888 Fax 095 564 8787 South Africa Johannesburg Tel 27 11 478 0411 Fax 27 11 478 0349 Spain Madrid Tel 1 806 1200 Fax 1 804 0414 Sweden Sundbyberg Tel 46 08 619 4400 Fax 46 08 619 4401 Switzerland Rotkreuz Tel 041 799 7708 Fax 041 790 0676 United Kingdom Warrington Cheshire Tel 01925 825650 Fax 01925 282502 Regional Sales Offices All Other European Countries Middle East West Asia Africa Except South Africa Langen Germany Tel 49 6103 708 301 Fax 49 6103 708 310 EASTERN ASIA CHINA OCEANIA PACIFIC RIM Australia Scoresby Victoria Tel 03 9212 8585 Fax 03 9212 8502 China Beijing Tel 86 10 6238 1156 Fax 86 10 6238 1162 Hong Kong Tel 852 2590 0238 Fax 852 2590 0513 Japan Chiba Tel 0473 80 8500 Fax 0473 80 8505 Korea Seoul Tel 822 592 7238 Fa
8. NG 1 U uL 0 50 50 Total Volume 15 75 1575 13 Plate Configuration for Primer Optimization 5 uM 5 uM PCR Master Forward Reverse Deionized Wells Mix uL Primer pL Primer uL Template Water pL A1 A4 15 75 0 5 0 5 5 0 28 25 A5 A8 15 75 0 5 3 0 5 0 25 75 A9 A12 15 75 0 5 9 0 5 0 19 75 B1 B4 15 75 3 0 0 5 5 0 25 75 B5 B8 15 75 3 0 3 0 5 0 23 25 B9 B12 15 75 3 0 9 0 5 0 17 25 C1 04 15 75 9 0 0 5 5 0 19 75 C5 C8 15 75 9 0 3 0 5 0 17 25 C9 C12 15 75 9 0 9 0 5 0 11 25 D1 D4 15 75 0 5 0 5 0 33 25 D5 D8 15 75 0 5 3 0 0 30 75 D9 D12 15 75 0 5 9 0 0 24 75 E1 E4 15 75 3 0 0 5 0 30 75 E5 E8 15 75 3 0 3 0 0 28 25 E9 E12 15 75 3 0 9 0 0 22 25 F1 F4 15 75 9 0 0 5 0 24 75 F5 F8 15 75 9 0 3 0 0 22 25 F9 F12 15 75 9 0 9 0 0 16 25 Thermal Cycling Parameters for Primer Optimization Times and Temperatures AmpErase UNG AmpliTag Gold Each of 40 Cycles Incubation Activation Melt Anneal Extend HOLD HOLD CYCLE 2 min 10 min 15 sec 1 min 50 C 95 C 95 C 60 C Determine the Optimal Primer Concentrations Step Action 1 Prepare a PCR master mix to run four replicates of each of the nine conditions as shown The master mix is described in PCR Master Mix for Primer Optimization on page 13 Reverse Forward Primer nM Primer nM 50 300 900 50 50 50 300 50 900 50 300
9. PCR process and the non specific detection format of SYBR Green chemistry make special laboratory practices necessary Small levels of DNA carryover from samples with high DNA concentrations from the DNA Template Controls or from previous PCR amplifications can result in product even in the absence of added template DNA Because the indicator dye will bind to any double stranded DNA unintended products will result in a positive signal See the references in Appendix B on page 20 for more information on the polymerase chain reaction and the prevention of unintended products To improve PCR specificity and sensitivity by controlling mispriming events the Hot Start technique was introduced Faloona et al 1990 Hot Start PCR is a simple modification of the original PCR process where the amplification reaction is started at an elevated temperature This was initially performed manually by adding an essential component of the reaction to the reaction mixture only after that mixture had been heated to an elevated temperature However this approach was often cumbersome and time consuming especially when using large numbers of samples Recently PE Applied Biosystems introduced a new PCR enzyme AmpliTaq Gold DNA Polymerase to perform an automated convenient and efficient Hot Start AmpliTaq Gold DNA Polymerase is a chemically modified form of AmpliTag9 DNA Polymerase The modification renders the enzyme inactive Upon thermal activatio
10. SYBR Green PCR and RT PCR Reagents Protocol PE Applied Biosystems DIVISION OF PERKIN ELMER Copyright 1998 The Perkin Elmer Corporation All rights reserved Products and procedures described in this protocol are intended for research purposes only Printed in the U S A Notice to Purchaser Limited License A license under U S Patents 4 683 202 4 683 195 and 4 965 188 or their foreign counterparts owned by Hoffmann La Roche Inc and F Hoffmann La Roche Ltd Roche for use in research and development has an up front fee component and a running royalty component The purchase price of the SYBR Green PCR Core Reagents P N 4304886 includes limited non transferable rights under the running royalty component to use only this amount of the product to practice the Polymerase Chain Reaction PCR and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up front fee component Rights to the up front fee component must be obtained by the end user in order to have a complete license These rights under the up front fee component may be purchased from Perkin Elmer or obtained by purchasing an authorized thermal cycler No right to perform or offer commercial services of any kind using PCR including without limitation reporting the results of purchaser s activities for a fee or ot
11. el electrophoresis Step Action 1 Load 12 15 uL of sample per well on an ethidium bromide stained 4 NuSieve 3 1 agarose gel FMC P N 54928 2 For PCR fragments 100 bp run the gel at 80 100 V for 45 60 minutes For PCR fragments 100 250 bp run the gel at 100 115 V for 1 1 5 hours 3 Run samples 1 3 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 Amplifying Custom Target Sequences for Quantitation Overview Identify Target Sequence and Amplicon Size Design Primers We recommend the following steps for the development of quantitative PCR assays Step Action See page 1 Install Primer Express Software 2 Identify Target Sequence and Amplicon Size 10 3 Design Primers 10 4 Order Reagents 11 5 Quantitate Primers 11 6 Perform RT Reactions for Two Step RT PCR 12 7 Optimize Primer Concentrations for Two Step 13 RT PCR For best results use Primer Express primer design software PE Applied Biosystems reagents and the universal thermal cycling parameters given on page 14 A target template is a DNA cDNA or plasmid containing the nucleotide sequence of interest Design primers to amplify short segments of DNA within the target sequence These short segments are called amplicons Shorter amplicons work the most efficiently consistent results are obtained for
12. her commercial consideration is hereby granted by implication or estoppel Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at PE Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 This product is sold under licensing arrangements between Perkin Elmer and Life Technologies Inc The purchase price of this product includes limited non transferable rights under U S Patent 5 035 996 and foreign equivalents owned by Life Technologies Inc to use only this amount of the product to practice the claims in said patents solely for activities of the purchaser within the field of research Further information on purchasing licenses under the above patents may be obtained by contacting the Director Corporate Development at Life Technologies Inc 9800 Medical Center Dr Rockville MD 20850 The SYBR Green dye is sold under license from Molecular Probes Inc ABI PRISM MicroAmp and Perkin Elmer are registered trademarks of The Perkin Elmer Corporation ABI Applied Biosystems MultiScribe PE PE Applied Biosystems and Primer Express are trademarks of The Perkin Elmer Corporation AmpliTaq AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Contents Introd c
13. ion results use the following Primer Express software P N 402089 for primer design PE Applied Biosystems reagents PE Applied Biosystems universal thermal cycling conditions Follow the instructions in Amplifying Custom Target Sequences for Quantitation on page 10 Normalization The Passive Reference is a dye included in the 10X SYBR Green PCR Buffer that does not participate in the PCR amplification The Passive Reference provides an internal reference to which the SYBR Green dsDNA complex signal can be normalized during data analysis Normalization is necessary to correct for fluorescent fluctuations caused by changes in concentration or volume Multicomponenting Multicomponenting is the term used to distinguish the contribution each individual dye makes to the fluorescence spectra The overlapping spectra from the Pure Dye components generate the composite spectrum This spectrum represents one fluorescence intensity reading from one well The ABI PRISM 7700 Sequence Detector should include in its Pure Dye spectra the SYBR Green and ROX spectra for multicomponenting data generated with the SYBR Green PCR Core Reagents To add SYBR Green to the Pure Dye spectra use the Sequence Detection Systems Spectral Calibration Kit P N 4305822 Follow the detailed instructions in AB PRISM 7700 User Bulletin 4 Generating New Spectra Components P N 4306234 R and AR Values Normalization is accomplished by dividing the
14. ix with 3 mM Mg forward primer and reverse primer concentrations and 5 uL of RNAse free water NTC wells template standard STND wells or unknown template UNKN wells 17 Quantitation of Gene expression can be measured by the quantitation of cDNA cDNA Relative to a converted from a messenger RNA corresponding to this gene relative to Calibrator Sample a Calibrator sample serving as a physiological reference In a typical experiment gene expression levels are studied as a function of either a treatment of cells in culture of patients or of tissue type The calibrator sample in each case is the cDNA from either the untreated cells or patients or a specific tissue type All quantitations are also normalized to an endogenous control such as 18S rRNA to account for variability in the initial concentration and quality of the total RNA and in the conversion efficiency of the reverse transcription reaction All amplicons in these determinations should follow the amplicon design criteria defined previously around the Primer Express software Refer to User Bulletin 2 P N 4303859 for additional information about relative quantitation The plate below shows a typical configuration for relative quantitation with SYBR Green reagents o SYBR Relative Quantitation B Sample Type NAC No Amplification C Thermal Cycler Conditions 7700 Single Reporter Sample Name Comment Replica
15. lied Biosystems P N 402089 MicroAmp9 Optical 96 Well Reaction Plate and Optical Caps PE Applied Biosystems P N 403012 MicroAmp Optical 96 Well Reaction Plate PE Applied Biosystems P N N801 0560 Table 2 User supplied Materials continued Item Source MicroAmp Optical Tubes PE Applied Biosystems P N N801 0933 MicroAmp Optical Caps PE Applied Biosystems P N N801 0935 Sequence Detection Systems Spectral Calibration Kit PE Applied Biosystems P N 4305822 TaqMan Reverse Transcription Reagents PE Applied Biosystems P N N808 0234 Unlabeled primers 40nm 0 2 uM 1 0uM PE Applied Biosystems P N 450005 P N 450004 P N 450021 Centrifuge with adapter for 96 well plate Major laboratory suppliers MLS Microcentrifuge MLS NuSieve 4 3 1 agarose gels for DNA lt 1 kb FMC BioProducts P N 54928 Polypropylene tubes MLS Pipettors positive displacement or MLS air displacement Pipette tips with filter plugs MLS Vortexer MLS Tris EDTA TE Buffer pH 8 0 MLS Disposable gloves MLS Technical Support To Reach Us On the World Wide Web To Reach Us by Telephone Fax or E mail Our Web site address is http www perkin elmer com ab We strongly encourage you to visit our Web site for answers to frequently asked questions and to learn more about our products You can als
16. lko 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 Hes 13 319 335 Faloona F Weiss S Ferre S and Mullis K 1990 Abstract 1019 Sixth International Conference on AIDS June 20 24 1990 San Francisco CA Gelfand D H and White T J 1990 Thermostable DNA Polymerases In Innis M A Gelfand D H Sninsky J J and White T J eds PCR Protocols A Guide to Methods and Applications Academic Press Inc San Diego CA pp 129 141 Higuchi R Dollinger G Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences Bio Technology 10 413 417 Kwok S 1990 Procedures to minimize PCR product carry over In Innis M A Gelfand D H Sninsky J J and White T J eds PCR Protocols A Guide to Methods and Applications Academic Press Inc San Diego CA pp 142 145 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Longo N Berninger N S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction In Wu R ed Methods in Enzymology Vol 155 Academic Press Inc San Diego CA pp 335 350 Orrego C 1990
17. mr x 0 3 cm x C 100 C 258uM 11 Perform RT Reactions for If you are working with RNA you will need to generate a cDNA by reverse transcription PCR RT PCR The procedure for generating Two Step RT PCR cDNA using the TaqMan Reverse Transcription Reagents P N N808 0234 is described below Refer to the TaqMan Gold RT PCR Kit Protocol P N 402876 for more information on RT PCR RT Reaction Mix Component Volume Tube uL Final Concentration 10X TaqMan RT Buffer 10 1X 25 mM MgClo 22 5 5 mM deoxyNTPs mixture 20 500 uM of each dNTP Random Hexamer 2 5 uM RNase Inhibitor 2 0 4 U uL MultiScribe Reverse 2 5 1 25 U uL Transcriptase 50 U uL RNA Sample RNase free H2O 38 5 10 pg 2 ug Total 100 a Random hexamers oligo d T 1g or sequence specific reverse primers can be used for primers of cDNA synthesis Thermal Cycling Parameters for RT Reactions Step Incubation Reverse Reverse Transcriptase Transcription Inactivation HOLD HOLD HOLD Time 10 min 30 min 5 min Temperature 25 C 48 C 95 C a If using random hexamers or oligo d T primers for first strand cDNA synthesis a primer incubation step is necessary at 25 C for 10 minutes Perform RT Reactions Step Action 1 Prepare the RT Reaction Mix by combining all the nonenzymatic components listed in RT Reaction Mix above 2 Vortex briefly Perform RT Reactions continued Step
18. n the modifier is released resulting in active enzyme The high temperature incubation step required for activation ensures that active enzyme is generated only at temperatures where the DNA is fully denatured When AmpliTaq Gold DNA Polymerase is added to the reaction mixture at room temperature the inactive enzyme is not capable of primer extension Any low stringency mispriming events that may have occurred will not be enzymatically extended and subsequently amplified AmpErase UNG General PCR Practices AmpliTaq Gold DNA Polymerase can be introduced into existing amplification systems with only minimal alterations to the reaction protocol resulting in higher specificity sensitivity and product yield AmpErase uracil N glycosylase UNG is a pure nuclease free 26 kDa recombinant enzyme encoded by the Escherichia coli uracil N glycosylase gene This gene has been inserted into an E coli host to direct expression of the native form of the enzyme Kwok and Higuchi 1989 UNG acts on single and double stranded dU containing DNA It acts by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The enzyme causes the release of uracil 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 AmpErase UNG treatment can prevent the reamplification of carryover PCR products When dUTP replaces dTTP in PCR amplification AmpErase UNG trea
19. o order technical documents and or an index of available documents and have them faxed to you through our site see the Fax on Demand section below Answers to Frequently Asked Questions http Awww2 perkin elmer com ab FAQ frontend html Phone 1 800 762 4001 Choose PCR or Sequence Detection Fax 1 203 761 2542 E mail pcrlab perkin elmer com Hours for n the United States and Canada technical support is available Telephone between 5 30 a m and 5 00 p m Pacific Time Technical Support Outside of the United States and Canada call your local sales office see the back cover of this protocol Fax on Demand Free 24 hour access to PE Applied Biosystems technical documents is available by fax There are two ways to access Fax on Demand documents Order through our Web site on the Internet at http www perkin elmer com fod Search for documents to order using keywords Up to five documents can be faxed to you if you already know the titles Order by phone Inthe United States and Canada call 1 800 487 6809 from a touch tone phone Outside the United States and Canada call 650 596 4419 from a touch tone phone Have your fax number ready Press 1 to order an index of available documents and have it faxed to you Press 2 to have up to five documents faxed to you Preventing Non Specific Amplification Overview Hot Start PCR AmpliTag Gold DNA Polymerase The DNA amplification capability of the
20. r Mix for Primer Optimization Volume per 50 uL Volume per Component Reaction uL 100 wells pL 10X SYBR PCR Buffer 5 0 500 25 mM MgCl 6 0 600 dNTP Blend 2 5 mM dATP 2 5 mM 6 0 600 dCTP 2 5 mM dGTP 5 0 mM dUTP AmpliTaq Gold 5 U uL 0 25 25 MultiScribe Reverse Transcriptase 0 25 25 50 U uL RNase Inhibitor 1 0 100 Total Volume 18 50 1850 Appendix A Absolute and Relative Quantitation Overview Two types of quantitation are possible with the SYBR Green reagents Relative quantitation of a target against an internal standard is particularly useful for gene expression measurements Absolute quantitation is possible if the isolation procedure and sample contents do not impact the PCR results The quantitation of genomic DNA may lend itself for absolute quantitation against a standard curve Absolute Absolute quantitation compares the C4 of an unknown sample against a Quantitation of standard curve with known copy numbers Absolute quantitation on the Plasmids or ABI PRISM 7700 Sequence Detector can be carried out with the plate Genomic DNA Configuration shown below NEEN _ Absolute Quantitation Sample Type _ Aies t Thermal Cycler Conditions 7700 Single Reporter Sample Name Comment Replicate Quantity Show Analysis Dye Layer SYER E Each well in this plate receives 45 uL of a master m
21. sk te pete EUER P EERUR ERU BE 11 Quantitate Primers 0 0 0 0 eee eens 11 Perform RT Reactions for Two Step RT PCR lusus 12 Optimize Primer Concentrations for Two Step RT PCR 13 Optimize Primer Concentrations for One Step RT PCR 16 Appendix A Absolute and Relative Quantitation 17 OV rVIeW ec eg oed tv a RSA Ried SRE ea ae eee 17 Absolute Quantitation of Plasmids or Genomic DNA 17 Quantitation of cDNA Relative to a Calibrator Sample 18 Appendix B References 1 45 A ae oes tek aves ER eS 20 Introduction Purpose Interpreting Results The SYBR Green PCR Core Reagents P N 4304886 are designed to detect genomic plasmid and cDNA with SYBR Green dye Direct detection of polymerase chain reaction PCR product is monitored by measuring the increase in fluorescence caused by the binding of SYBR Green to double stranded ds DNA In RNA quantitation assays the SYBR Green PCR Core Reagents are used in the second step of a two step reverse transcription polymerase chain reaction RT PCR protocol The template in this case is the cDNA generated from a reverse transcription reaction The SYBR Green PCR Core Reagents are designed for use with the ABI Prism 7700 Sequence Detector The excitation emission profile for the SYBR Green dye is similar to that of the FAM dye This allows the use of the ROX Passive Reference molecule For the best quantitat
22. te Quantity Show Analysis Dye Layer Sample 1 i Sample 1 i Sample 1 Sample 1 Sample Sample 2 T2 T2 T2 T2 T2 T2 Sample 4 i Sample 4 i Sample 4 Sample 4 Sample 5 Sample 5 TE i I TES dere Sample 1 Sample 1 i Sample In this configuration endogenous controls are run for each sample in rows A and B and targets T1 T3 are run on rows C H Each row shows wells corresponding to a calibrator sample and up to five experimental 18 samples All wells are run in quadruplicate The analysis of relative quantitation for a target template in samples 1 5 requires the following The mean C value of the replicate wells run for each sample The difference ACT between the mean Cy values of the samples in the target wells T1 T3 and those of the endogenous controls in the ENDGNS wells The difference AACT between the ACT values of the samples for each target and the mean C value of the Calibrator for that target AAC The relative quantitation value is expressed as 2 m A semilog plot of these values provides a graphical representation of the relative quantitation values obtained in this plate 19 Appendix B References 20 Ausubel F M Brent R Kingstin R E Moore D D Seidman J G Smith J A and Struhl K eds 1987 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience John Wiley and Sons New York Delort A M Duplaa A M Mo
23. tions l c esee eue EES E ORO OS EAE ES eo vs 1 P rposeca vg tens tere ue pe edet RUNS SEE UT REUS ES 1 Interpreting Results 0 0 0 cece eee eens 1 Real Time Detection 0 0 a cette 3 Materials and Equipment 1 0 0 0 cece eee eee 4 Kit Contents oes a cae eh ce edb A REGE 4 Storage and Stability llle 4 MSDS ax ite ac RADI RA ER RE CURE E 4 Materials Required But Not Supplied 00 0 4 Technical Support conse decane eda gee te D Mere dts dere bs 6 To Reach Us On the World Wide Web 0000 6 To Reach Us by Telephone Fax or E mail 6 Hours for Telephone Technical Support 0 0 6 Fax on D emand 3 iss shi tak ea Wala Re a Rea woes S 6 Preventing Non Specific Amplification llle ee eee 7 OVERVIEW 226 otl edes d tede eR dei eR NEU CR EIU NES tnt 7 Hot Start PCR Rache er pa e RE 7 AmpliTaq Gold DNA Polymerase 00000000005 7 AmpEraseUNG x si Soak poh ER Ch ess bee hee or 8 General PCR Practices 0 0 eee eee eee 8 Fluorescent Contaminants 0 0 0 0 ee eee eee eee 9 Agarose Gels to Check PCR Product Purity 9 Amplifying Custom Target Sequences for Quantitation 10 OVELVIEW 2 ssi ets Soe Gee Ses ta E eee a ee 10 Identify Target Sequence and Amplicon Size 10 Design Prmers poi eke WA ogee nex RR e 10 Order Reagents iis3 31 cae
24. tment can remove up to 200 000 copies of amplicon per 50 uL reaction Please follow these recommended procedures Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Use a positive displacement pipet or aerosol resistant pipet tips Clean lab benches and equipment periodically with 1096 bleach solution Fluorescent Contaminants Agarose Gels to Check PCR Product Purity Since fluorescent contaminants can interfere with this assay and give false positive results it may be necessary to include a No Amplification Control tube that contains sample but no enzyme If the absolute fluorescence of the No Amplification Control is greater than that of the No Template Control after PCR fluorescent contaminants may be present in the sample or in the heat block of the thermal cycler The absence of non specific amplification can be confirmed by analyzing the PCR amplification products by agarose g
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