Agilent Technologies Brilliant III Ultra-Fast SYBR Green QPCR Master Mix Instruction Manual
Contents
1. kod 22 2 a 21 20 19 18 7 16 15 14 13 0 0001 0 001 ao am 01 a2 1 2345 10 290 109 200 1000 65 0 70 0 75 0 80 0 85 0 90 0 95 0 Quantity Tm 83 76 Temperature C Figure 1 Top panel StepOnePlus instrument amplification plot A serial dilution of cDNA template was added in quadruplicate to each reaction The fluorescence value used to determine Ct the threshold line is shown as a solid line Bottom left panel Standard curve generated from amplification plot An amplification efficiency of 102 56 and an R squared value of 0 998 were obtained Bottom right panel Melt curve generated when amplified products were subjected to dissociation analysis The fluorescence peaks corresponding to the amplicon are centered around 83 76 C Brilliant Ill Ultra Fast SYBR Green QPCR Master Mix 3 PREPROTOCOL CONSIDERATIONS PCR Primers Reference Dye It is critical in SYBR Green based QPCR to minimize the formation of non specific amplification products This issue becomes more prominent at low target concentrations Therefore to maximize the sensitivity of the assay it is necessary to use the lowest concentration of primers possible without compromising the efficiency of PCR It is important to consider both the relative concentrations of forward and reverse primers and the total primer concentration The optimal concentration of the upstream and downstream PCR primers is the lowest concentration that results in the lowest Ct an2. agilent com Brilliant III Ultra Fast SYBR Green QPCR Master Mix CONTENTS Materials Provided sccscscsssssessecssssesssssssesssrssessecsscssessecsscseesssssnecsassonsseesecssnssesscssaseosesecsessonseeese Storage Conditions ceissssssssscssssscsasessss cosvesscensecesssssvessoocscssecesevasoonsdessecssceasesedacssecsssensssesxasdesseseebessanasccaxs Additional Materials Required seessesssesssesssesssecsseosseossoossoossoossoossoossoossosssosssossssssssesssesssesssesssessssse Notices To Purchaser cccccsssssssceccsssesssssssecsscssescecsscsssssecsscssssenecsessonssesecssnssessecsscssssssecsecsasssnseeese IS AAA _ ERAS Fluorescence Monitoring in Real Time Using SYBR Green oooococonococicoccncncoronononononrnnononos Preprotocol Considerations cccscsccscsccsecscsccsccccssccecssscecsccsescccsesccesscesssccsescccsescesssssessscsseseseesees PCR Primer ita eros Reference Dy eiiceiorsteiesicinceltivisdort sable vedovs dhe italia italia Magnesium Chloride viv oia ii Data Acquisition with a Spectrofluorometric Thermal Cycler oooonnccnnccnocicocaconaconncannananannss Multiplex PER AA eee tin Preventing Template Cross Contamination 0 0 00 ceceeeeeseeeseeeseeeseecnsecsaeceaecnseesseeeseeeseeseneeees Protocol ASTE E T Preparing the REACH ONS iris ai odd Sova r E E T E EE PCR Cycling Pro TS sidene ene drid Dissociation Pro Stams esena a a e Coe E EE EE E A O aE S Troubleshooting si cices vests s
3. at 4 C for up to three months or returned to 20 C for long term storage SYBR Green dye present in the master mix is light sensitive solutions containing the master mix should be protected from light whenever possible 1 If using the reference dye dilute the provided dye with nuclease free PCR grade H2O For the ABI StepOnePlus instrument or the ABI 7900HT Fast instrument dilute the dye 1 50 for a final concentration of 300 nM in the reactions For the Stratagene Mx3000P or Mx3005P instrument or the ABI 7500 Fast instrument dilute the dye 1 500 for a final concentration of 30 nM in the reactions Keep all solutions containing the reference dye protected from light 2 Prepare the experimental reactions by adding the following components in order Prepare a single reagent mixture for multiple reactions using multiples of each component listed below Reagent Mixture Nuclease free PCR grade HzO to adjust the final volume to 20 ul including experimental DNA 10 ul of 2x SYBR Green QPCR master mix x ul of upstream primer 200 500 nM final concentration is recommended x ul of downstream primer 200 500 nM final concentration is recommended 0 3 ul of diluted reference dye from step 1 optional 3 Gently mix without creating bubbles bubbles interfere with fluorescence detection do not vortex then distribute the mixture to individual PCR reaction tubes 4 Add x ul of experimental gDNA cDNA or plasmid DNA to each reaction to
4. Brilliant IIl Ultra Fast SYBR Green QPCR Master Mix Instruction Manual Catalog 600882 single kit 600883 10 pack kit Revision B Research Use Only Not for Use in Diagnostic Procedures 600882 12 oie Agilent Technologies LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Agilent Agilent shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product ORDERING INFORMATION AND TECHNICAL SERVICES United States and Canada Agilent Technologies Stratagene Products Division 11011 North Torrey Pines Road La Jolla CA 92037 Telephone 858 373 6300 Order Toll Free 800 424 5444 Technical Services 800 894 1304 email techservices agilent com World Wide Web www genomics agilent com Europe Location Telephone Austria 01 25125 6800 Benelux 02 404 92 22 Denmark 45 70 13 00 30 Finland 010 802 220 France 0810 446 446 Germany 0800 603 1000 Italy 800 012575 Netherlands 020 547 2600 Spain 901 11 68 90 Sweden 08 506 4 8960 Switzerland 0848 8035 60 UK Ireland 0845 712 5292 All Other Countries Please contact your local distributor A complete list of distributors is available at www genomics
5. bring the final reaction volume to 20 ul 5 Gently mix the reactions without creating bubbles do not vortex then centrifuge the reactions briefly 6 Place the reactions in the instrument Based on the instrument you are using select the appropriate PCR program from the tables below Set the instrument to detect and report fluorescence at each cycle during the 60 C annealing extension step ABI 7500 Fast ABI 7900HT Fast Cycles Duration of cycle Temperature 1 3 minutes 95 C 40 5 seconds 95 C 12 seconds 60 C ABI StepOnePlus Cycles Duration of cycle Temperature 1 3 minutes 95 C 40 5 seconds 95 C 10 seconds 60 C QIAGEN Rotor Gene Q Cycles Duration of cycle Temperature 1 3 minutes 95 C 40 10 seconds 95 C 10 20 seconds 60 C Stratagene Mx3000P and Mx3005P Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 seconds 95 C 15 seconds 60 C Bio Rad CFX96 Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 seconds 95 C 5 10 seconds 60 C Roche LightCycler 480 Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 seconds 95 C 10 seconds 60 C Cycles Duration of cycle Temperature 1 3 minutes 95 C 40 5 20 seconds 95 C 20 seconds 60 C 7 For your specific instrument follow the manufacturer s guidelines for generati
6. d an adequate fluorescence for a given target concentration with minimal or no formation of primer dimer This concentration should be determined empirically Generally primer concentrations in the range of 200 500 nM are satisfactory A passive reference dye is included in this kit and may be added to compensate for non PCR related variations in fluorescence Fluorescence from the passive reference dye does not change during the course of the PCR reaction but provides a stable baseline to which samples are normalized In this way the reference dye compensates for changes in fluorescence between wells caused by slight volume differences in reaction tubes The excitation and emission wavelengths of the reference dye are 584 nm and 612 nm respectively Although addition of the reference dye is not required when using the Bio Rad CFX96 real time PCR system with other instruments including the ABI StepOnePlus instrument the use of the reference dye may be required for optimal results Reference Dye Dilution Recommendations Prepare fresh dilutions of the reference dye prior to setting up the reactions and keep all tubes containing the reference dye protected from light as much as possible Make initial dilutions of the reference dye using nuclease free PCR grade H O If using a StepOnePlus or 7900HT Fast instrument dilute the dye 1 50 for a final concentration of 300 nM in the reactions For the Stratagene Mx instruments or the ABI 7500 Fast
7. fluorescence detection Brilliant 111 Ultra Fast SYBR Green QPCR Master Mix PCR Cycling Programs Place the reactions in the instrument Based on the instrument you are using select the appropriate PCR program from the tables below Set the instrument to detect and report fluorescence at each cycle during the 60 C annealing extension step ABI 7900HT Fast 6 ABI 7500 Fast Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 seconds 95 C 12 seconds 60 C ABI StepOnePlus Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 seconds 95 C 10 seconds 60 C QIAGEN Rotor Gene Q Cycles Duration of cycle Temperature 1 3 minutes 95 C 40 10 seconds 95 C 10 20 seconds 60 C Cycles Duration of cycle Temperature 1 3 minutes 95 C 40 5 seconds 95 C 15 seconds 60 C Bio Rad CFX96 Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 seconds 95 C 5 10 seconds 60 C Roche LightCycler 480 Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 seconds 95 C 10 seconds 60 C Stratagene Mx3000P and Mx3005P Cycles Duration of cycle Temperature 3 minutes 95 C 40 5 20 seconds 99G 20 seconds 60 C Dissociation Programs 7 For your specific instrument follow the manufacturer s guidelines for generating dissociation cu
8. instrument dilute the dye 1 500 for a final concentration of 30 nM The Bio Rad CFX96 the Roche LightCycler 480 and the QIAGEN Rotor Gene Q instruments do not require the use of the reference dye The diluted reference dye if stored in a light protected tube at 4 C can be used within the same day for setting up additional assays Brilliant 111 Ultra Fast SYBR Green QPCR Master Mix Magnesium Chloride The optimal MgCl concentration promotes maximal amplification of the specific target amplicon with minimal nonspecific products and primer dimer formation High levels of the Mg ion tend to favor the formation of nonspecific dsDNA including primer dimers Therefore when a SYBR Green based QPCR assay is being optimized the MgCl levels should be as low as possible without compromising the efficiency of amplification of the specific target typically between 1 5 and 2 5 mM MgCl The Brilliant II Ultra Fast SYBR Green QPCR master mix contains MgCl at a concentration of 2 5 mM in the 1x solution which is suitable for most targets The concentration may be increased if desired by adding a small amount of a concentrated MgCl solution to the 1x experimental reaction at the time of setup Data Acquisition with a Spectrofluorometric Thermal Cycler The instrument should be set to collect SYBR Green I data in real time at the annealing extension step of each cycle How this is accomplished will depend on the software that comma
9. nds the particular instrument you are using Consult the manufacturer s instruction manual for the instrument and software version you are using Multiplex PCR Multiplex PCR is the amplification of more than one target in a single polymerase chain reaction Because SYBR Green I dye fluoresces in the presence of any dsDNA multiplexing with the Brilliant III Ultra Fast SYBR Green QPCR master mix is not recommended Preventing Template Cross Contamination Take precautions to minimize the potential for carryover of nucleic acids from one experiment to the next Use separate work areas and pipettors for pre and post amplification steps Use positive displacement pipets or aerosol resistant pipet tips Brilliant Ill Ultra Fast SYBR Green QPCR Master Mix 5 PROTOCOL Preparing the Reactions Notes Once the tube containing the 2x QPCR master mix is thawed store it on ice while setting up the reactions Following initial thawing of the master mix store the unused portion at 4 C for up to three months or return to 20 C for long term storage SYBR Green dye is light sensitive solutions containing the master mix should be protected from light whenever possible It is prudent to set up a no template control reaction to screen for amplicon contamination or false amplification If using the reference dye dilute the provided dye using nuclease free PCR grade H O For the ABI StepOnePlus instrument or the ABI 7900HT Fast i
10. ng dissociation curves The exact duration needs to be optimized for each target Genomic targets generally require longer denaturation and annealing extension times than low complexity targets e g cDNA and plasmid DNA
11. ng this reagent separately allows the user to control the final dye concentration increasing the flexibility of the reagents for use with multiple platforms Fluorescence Monitoring in Real Time Using SYBR Green When fluorescence signal from a SYBR Green based PCR reaction is monitored in real time the results can be displayed as an amplification plot which reflects the change in fluorescence during cycling Studies have shown that initial copy number can be quantitated during real time PCR analysis based on threshold cycle Ct Ct is defined as the cycle at which fluorescence is determined to be statistically significant above background In the amplification plot in Figure 1 for example the Ct of each reaction is the cycle number at which the plot crosses the threshold line The threshold cycle has been shown to be inversely proportional to the log of the initial copy number The more template that is initially present the fewer the number of cycles it takes to get to a point where the fluorescence signal is detectable above background Quantitative information based on threshold cycle is more accurate than information based on endpoint determinations as it is based on measurements taken during the exponential phase of PCR amplification when the PCR efficiency has yet to be influenced by limiting reagents small differences in reaction components or cycling conditions In Figure 1 the Brilliant III Ultra Fast SYBR Green QPCR master mi
12. nstrument dilute the dye 1 50 for a final concentration of 300 nM in the reactions For the Stratagene Mx3000P or Mx3005P instrument or the ABI 7500 Fast instrument dilute the dye 1 500 for a final concentration of 30 nM in the reactions Keep all solutions containing the reference dye protected from light Prepare the experimental reactions by combining the following components in order Prepare a single reagent mixture for replicate experimental reactions and replicate no template controls plus at least one reaction volume excess using multiples of each component listed below Reagent Mixture Nuclease free PCR grade water to adjust the final volume to 20 ul including experimental DNA 10 ul of 2x SYBR Green QPCR master mix x ul of upstream primer 200 500 nM final concentration x ul of downstream primer 200 500 nM final concentration 0 3 ul of diluted reference dye optional Gently mix without creating bubbles do not vortex then distribute the mixture to individual PCR reaction tubes Add x ul of experimental DNA to each reaction to bring the final reaction volume to 20 ul The table below lists a suggested quantity range for different DNA templates DNA Quantity per reaction Genomic DNA 5 pg 50 ng cDNA 0 5 pg 100 ng Refers to RNA input amount during cDNA synthesis Gently mix the reactions without creating bubbles do not vortex then centrifuge the reactions briefly Note Bubbles interfere with
13. on or by estoppel Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Notice to Purchaser Limited License SYBR Green is licensed for research and development only under patents and patent applications owned by Invitrogen Corporation Revision B Agilent Technologies Inc 2010 Brilliant Ill Ultra Fast SYBR Green QPCR Master Mix INTRODUCTION The Brilliant III Ultra Fast SYBR Green QPCR Master Mix is a single tube reagent designed for performing accelerated quantitative PCR amplifications on the ABI StepOnePlus and Bio Rad CFX96 real time PCR instruments and other fast cycling systems such as the ABI 7900HT and 7500 Fast systems The master mix includes two key components that enable it to perform optimally under fast cycling conditions e A mutated form of Tag DNA polymerase that has been specifically engineered for faster replication e Animproved chemical hot start mechanism that promotes faster hot start release to improve amplification specificity while keeping the run time of the PCR protocol to a minimum The 2x master mix contains the mutant Tag DNA polymerase dNTPs Mg a buffer specially formulated for fast cycling and the double stranded DNA binding dye SYBR Green I for detection A passive reference dye an optional reaction component is provided in a separate tube Providi
14. osssssesasecsessossenscensecesssssbessosnsecescesvestoovsscedscesuvadesesncesecsssencesusdedvessvestscossseseness A NON Brilliant IIl Ultra Fast SYBR Green QPCR Master Mix MATERIALS PROVIDED Catalog 600882 single kit 600883 10 pack kit Materials Provided Quantity 2x Brilliant Ill Ultra Fast SYBR Green QPCR Master Mix 2x2 ml Reference dye 1 mM 100 ul a Sufficient PCR reagents are provided for four hundred 20 ul reactions gt Quantities listed are for a single kit For 10 pack kits each item is provided at 10 times the listed quantity The master mix contains nucleotide mix GATC d The master mix and reference dye are light sensitive and should be kept away from light whenever possible STORAGE CONDITIONS All Components Store at 20 C upon receipt After thawing the 2x master may be stored at 4 C for up to three months or returned to 20 C for long term storage Note The SYBR Green master mix and the reference dye are light sensitive and should be kept away from light whenever possible ADDITIONAL MATERIALS REQUIRED Spectrofluorometric thermal cycler Nuclease free PCR grade water NOTICES TO PURCHASER Notice to Purchaser Limited License Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser s own internal research No other patent rights are conveyed expressly by implicati
15. rves The exact duration needs to be optimized for each target Genomic targets generally require longer denaturation and annealing extension times than low complexity targets e g cDNA and plasmid DNA Brilliant Ill Ultra Fast SYBR Green QPCR Master Mix TROUBLESHOOTING Observation No or little increase in Suggestion s Optimize the primer concentration fluorescence with cycling The target is highly GC rich Raise the denaturation temperature to 98 C or titrate DMSO into the reactions in 1 increments Ensure that the correct concentration and amount of template was used and that the emplate sample is of good quality If unsure make new serial dilutions of template before repeating PCR It may also be possible to check for PCR inhibitors by adding this arget into an assay that is known to work Use a sufficient number of cycles in the PCR reaction Gel analyze PCR product to determine if there was successful amplification Ensure the correct dilution of reference dye was used The DNA polymerase was not activated Ensure that the 3 minute initial incubation at 95 C was performed as part of the cycling parameters The DNA polymerase was activated for more than 3 minutes Ensure that the initial 95 C incubation was not longer than 3 minutes The MgCl concentration is not optimal The MgCl concentration in the 1x Brilliant IIl Ultra Fast SYBR Green QPCR master mix is 2 5 mM It i
16. s possible to add small amounts of concentrated MgCl to the experimental reactions to increase the MgCl concentration if desired Target length may be too long for sufficient amplification with fast cycling Design the primers so that the PCR product is lt 300 bp in length There is a large abundance of primer dimer and nonspecific PCR products Reduce primer concentrations or re design primers REFERENCES 1 Higuchi R Fockler C Dollinger G and Watson R 1993 Biotechnology N Y 11 9 1026 30 2 Edwards M and Gibbs R 1995 Multiplex PCR In PCR Primer A Laboratory ENDNOTES Manual C W Dieffenbach and G S Dveksler Eds pp 157 171 Cold Spring Harbor Laboratory Press Plainview NY LightCycler is a registered trademark of Roche SYBR is a registered trademark of Molecular Probes Inc MSDS INFORMATION The Material Safety Data Sh eet MSDS information for Stratagene products is provided on the web at http www genomics agilent com MSDS documents are not included with product shipments Brilliant Ill Ultra Fast SYBR Green QPCR Master Mix STRATAGENE An Agilent Technologies Division BRILLIANT III ULTRA FAST SYBR GREEN QPCR MASTER MIX Catalog 600882 600883 QUICK REFERENCE PROTOCOL Prior to setting up the reactions thaw the 2x SYBR Green QPCR master mix and store on ice Following initial thawing of the master mix the unused portion may be stored
17. x was used in reactions containing serially diluted cDNA template 0 5 pg 50 ng and a no template control reaction NTC to amplify the GAPDH target In the amplification plot top panel the reactions containing template show a significant increase in fluorescence with Ct values ranging from 17 to 33 The NTC reaction has no Ct because the amplification plot does not cross the threshold The Ct values obtained in the amplification plot are used to generate the standard curve in the bottom left panel In the dissociation melt curve bottom right panel PCR samples were subjected to a stepwise increase in temperature from 60 C to 95 C with fluorescence measurements taken throughout this range The first derivative of fluorescence is then plotted versus temperature As the temperature increases the amplification Brilliant 111 Ultra Fast SYBR Green QPCR Master Mix products in each tube melt according to their composition In Figure 1 the melt curve shows fluorescence peaks centered at 83 76 C which correspond to the desired product in the reactions If primer dimer or nonspecific products had been made during the amplification step they would generally melt at a lower temperature defined as the Tm than the desired products Amplification Plot 2 4 6 a 10 12 14 16 181MU20NM 2 A BD B XX DL M A KB 4 Cycle Standard Curve Melt Curve 34 33 32 31 30 29 28 27 a c 26 25 3 2 i 24 e o v 23 2
Download Pdf Manuals
Related Search
Agilent Technologies Brilliant III Ultra Fast SYBR Green QPCR Master Mix Instruction Manual