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FullVelocity™ SYBR® Green QPCR Master Mix INSTRUCTION MANUAL Catalog #600581 (single kit) Catalog #929581 (10-pack kit) Revision #056003a For In Vitro Use Only *600581-12_056003a/* 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 Stratagene. Stratagene 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 Stratagene 11011 North Torrey Pines Road La Jolla, CA 92037 Telephone (858) 373-6300 Order Toll Free (800) 424-5444 Technical Services (800) 894-1304 Internet [email protected] World Wide Web www.stratagene.com Stratagene European Contacts Location Telephone Fax Technical Services Austria 0800 292 499 0800 292 496 0800 292 498 Belgium France Germany Netherlands Switzerland United Kingdom 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 15775 0800 15740 0800 15720 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 919 288 0800 919 287 0800 919 289 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 182 8232 0800 182 8231 0800 182 8234 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 023 0446 +31 (0)20 312 5700 0800 023 0448 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 563 080 0800 563 082 0800 563 081 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 917 3282 0800 917 3283 0800 917 3281 All Other Countries Please contact your local distributor. A complete list of distributors is available at www.stratagene.com. FullVelocity™ SYBR® Green QPCR Master Mix CONTENTS Materials Provided.............................................................................................................................. 1 Storage Conditions .............................................................................................................................. 1 Additional Materials Required .......................................................................................................... 1 Notices to Purchaser ........................................................................................................................... 1 Introduction......................................................................................................................................... 2 Features of Kit Components.................................................................................................. 2 Passive Reference Dye .......................................................................................................... 3 SYBR® Green I Dye.............................................................................................................. 3 Faster QPCR using the FullVelocity™ SYBR® Green QPCR Master Mix .......................... 3 Fluorescence Monitoring in Real-Time................................................................................. 4 Preprotocol Considerations................................................................................................................ 6 cDNA Synthesis and RNA Isolation ..................................................................................... 6 Primer Design and Concentration ......................................................................................... 6 Reference Dye ....................................................................................................................... 7 Magnesium Chloride Concentration...................................................................................... 7 Preparing a Single Reagent Mixture for Multiple Samples................................................... 7 Mixing and Pipetting Enzyme Solutions ............................................................................... 7 Recommended Control Reactions ......................................................................................... 8 Preventing Template Cross-Contamination........................................................................... 8 Fluorescence Detection and Data Acquisition....................................................................... 8 Protocols............................................................................................................................................... 9 Preparing the Reactions......................................................................................................... 9 PCR Cycling Program ......................................................................................................... 10 Dissociation Program .......................................................................................................... 11 Troubleshooting ................................................................................................................................ 12 References .......................................................................................................................................... 13 Endnotes............................................................................................................................................. 13 MSDS Information............................................................................................................................ 13 Quick-Reference Protocol ................................................................................................................ 15 FullVelocity™ SYBR® Green QPCR Master Mix MATERIALS PROVIDED Catalog #600581 (single kit) and Catalog #929581 (10-pack kit) Materials provided Quantitya, b 2× FullVelocity™ SYBR® Green QPCR Master Mix 2 × 2.5 ml Reference dye, 1 mM a b 100 μl Sufficient PCR reagents are provided for four hundred, 25-μl reactions. Catalog #929581 contains 10 Catalog #600581 kits. STORAGE CONDITIONS All Components: Upon receipt, store all components at –20°C. Store the 2× master mix at 4°C after thawing. Once thawed, full activity of the master mix is guaranteed for 6 months. Note The reference dye and the 2× FullVelocity™ SYBR® Green QPCR master mix are lightsensitive and should be kept away from light whenever possible. ADDITIONAL MATERIALS REQUIRED Spectrofluorometric thermal cycler Nuclease-free PCR-grade water NOTICES TO PURCHASER Limited Label License No rights are granted to the purchaser hereunder to sell, modify for resale or otherwise transfer this product. Stratagene reserves all other rights, and this product may not be used in any manner other than as provided herein. Other Notices Use of certain labeling reagents may require licenses from entities other than Stratagene. Revision #056003a FullVelocity™ SYBR® Green QPCR Master Mix Copyright © 2006 by Stratagene. 1 INTRODUCTION ® The FullVelocity™ SYBR Green QPCR master mix kit provides rapid, economical real-time quantification of DNA and cDNA targets. FullVelocity technology is powered by a novel archaeal DNA polymerase specifically engineered for high speed amplification. Forty-cycle run times are reduced by up to 40% compared to conventional QPCR reagents, depending on the QPCR instrument platform used. The FullVelocity SYBR Green QPCR master mix includes a thermostable accessory protein that destabilizes mis-annealed primers at each cycle and neutralizing hot-start monoclonal antibody that prevents extension of mis-annealed primers during room temperature reaction set-up. Together, these specificity enhancers reduce interference from primer-dimers and spurious amplification products. The accelerated run times, reliability, specificity, and master mix format make the FullVelocity system an ideal choice for high-throughput QPCR applications. The FullVelocity SYBR Green QPCR master mix is easy to use and compatible with most existing primer-template systems, although some primer redesign may be necessary to optimize specificity or amplicon size (90–300 bp amplicons are recommended). The kit has been successfully used to amplify and detect a variety of high-, medium- and low-abundance targets from genomic DNA, plasmid DNA, and cDNA. The FullVelocity reagents and protocols have been optimized for maximum performance on Stratagene’s Mx4000®, Mx3000P®, and Mx3005P™ instruments, and may also be adapted for use on most other QPCR platforms. Features of Kit Components The 2× FullVelocity SYBR Green QPCR master mix includes a novel archaeal DNA polymerase specifically engineered for high-speed QPCR. This DNA polymerase exhibits a number of superior features including extreme thermostability, lack of 3´-5´ and 5´-3´ nuclease activities, and a specifically engineered DNA binding domain. In addition to the DNA polymerase, the 2× FullVelocity master mix includes an optimized PCR buffer, SYBR green I dye, MgCl2, nucleotides (GAUC), stabilizers, neutralizing hot start monoclonal antibodies, and a thermostable accessory protein that destabilizes mis-annealed primers and minimizes interference from non-specific amplification products. Stratagene’s expertise with archaeal enzymes has been used to produce a unique enzyme formulation that supports shorter denaturation and annealing/extension steps and tolerates temperature changes better than Taq DNA polymerase. 2 FullVelocity™ SYBR® Green QPCR Master Mix Passive Reference Dye The passive reference dye (with excitation and emission wavelengths of 584 nm and 612 nm, respectively) is provided as an optional reagent that may be added to compensate for non-PCR related variations in fluorescence. Providing the reference dye in a separate tube makes the FullVelocity SYBR Green QRT-PCR master mix adaptable for many real-time QPCR platforms (see Reference Dye in Preprotocol Considerations for more information). SYBR® Green I Dye The SYBR Green I dye1 has a high binding affinity to the minor groove of double-stranded DNA (dsDNA). It has an excitation maximum at 497 nm and an emission maximum at 520 nm. In the unbound state the dye exhibits little fluorescence; however, when bound to dsDNA, the fluorescence greatly increases, making it useful for the detection of product accumulation during real-time PCR. During denaturation, all DNA becomes singlestranded. At this stage, SYBR Green is free in solution and produces little fluorescence. During the annealing/extension step, the primers hybridize to the target sequence and are extended, resulting in dsDNA to which SYBR Green I can bind. The increase in fluorescence signal intensity over 40 amplification cycles depends on the initial concentration of target present in the PCR reaction. An important consideration when using SYBR Green I, however, is that it can bind to nonspecific dsDNA (e.g. primer-dimers and spurious PCR products) and fluoresce. Therefore, in the early stages of assay optimization, it is recommended that the PCR products are analyzed on a gel to verify production of the specific product of interest and to verify a correlation between the gel appearance and the fluorescence data. With every experiment, no-template controls should be performed to test for reagent contamination and primer-dimer formation. In addition, dissociation curve analysis is essential for discriminating between the synthesis of intended PCR product and the formation of primer-dimers or spurious amplification products. In general, careful primer design, optimizing primer concentration, and using primers of higher purity (e.g. HPLC-purified primers) can minimize the occurrence of side-reaction products, leading to more reliable DNA quantification. Faster QPCR using the FullVelocity™ SYBR® Green QPCR Master Mix The FullVelocity SYBR Green master mix has been specifically optimized for use with faster cycling times. For optimal sensitivity and specificity, it is critical that researchers modify their existing cycling regimen to shorten denaturation and annealing/extension times. See the PCR Cycling Program section of Protocols for the recommended cycling conditions for the FullVelocity SYBR master mix on various QPCR platforms. FullVelocity™ SYBR® Green QPCR Master Mix 3 Fluorescence Monitoring in Real-Time When fluorescence signal from a PCR reaction is monitored in real-time, the results can be displayed as an amplification plot (Figure 1), which reflects the change in fluorescence during cycling. This information can be used to quantitate initial copy number based on threshold cycle (Ct).2 Ct is defined as the cycle at which fluorescence is statistically significant above background. The threshold cycle is inversely proportional to the log of the initial copy number.2 The more template that is initially present, the fewer cycles required to reach the point where the fluorescence signal is detectable above background. Quantitative information based on threshold cycle is more accurate than information based on endpoint determinations because Ct is based on measurements taken during the exponential phase of PCR amplification when the PCR efficiency is not yet influenced by limiting reagents and small differences in reaction components or cycling conditions. Quantitative assessments based on endpoint fluorescence values (a single reading taken at the end of the PCR reaction) are inherently inaccurate because endpoint values can be greatly influenced by these factors. Figure 1 shows Mx3000P instrument amplification and dissociation curve plots obtained from amplifying a 550 bp amplicon in the absence (–template) or presence (+template) of template DNA. The reaction containing template shows a significant increase in fluorescence and has a Ct value ≅ 23. The reaction without template has a Ct of ≅37. To determine if the fluorescence in the no-template control reaction arises from true amplification of contaminating template in the reaction (primer stocks, master mix) or from the synthesis of primer-dimers or other non-specific products, a dissociation profile is generated. In the dissociation curve, PCR samples are subjected to a stepwise increase in temperature from 55°C to 95°C and fluorescence measurements are taken at every temperature increment. The melting of products causes SYBR Green dissociation, resulting in decreased fluorescence. After completion of the dissociation segment, fluorescence is plotted versus temperature. To simplify interpretation, the first derivative [–R´(T) or –Rn´(T)] should be plotted. As the temperature increases, the amplification products melt according to their composition. If primer-dimer or nonspecific products were made during the amplification step, they will generally melt at a lower temperature (Tm) than the desired products. The dissociation curve plot of these samples shows two fluorescence peaks: one in the “– template” reaction centered around 79°C (which corresponds to primer-dimer); and the other, in the “+ template” reaction, centered around 87°C (which corresponds to amplicon). In this way, the dissociation curve analysis of PCR products amplified in the presence of SYBR Green I dye can be a very powerful tool in the interpretation of fluorescence data. 4 FullVelocity™ SYBR® Green QPCR Master Mix + template – template + template – template Figure 1 Mx3000P multiplex quantitative PCR instrument amplification and dissociation curve plot of a reaction with and without template DNA. When the amplified products are subjected to dissociation curve analysis, the fluorescence peak corresponding to the amplicon (centered around 87°C) is distinguishable from the peak due to primer-dimer (centered around 79°C). FullVelocity™ SYBR® Green QPCR Master Mix 5 PREPROTOCOL CONSIDERATIONS cDNA Synthesis and RNA Isolation High-quality cDNA is essential for successful amplification with the FullVelocity SYBR Green QPCR master mix. Total and poly(A)+ RNA can be rapidly isolated and purified using Stratagene's Absolutely RNA® isolation kits, which are available for nano-, micro- and miniprep scale RNA purifications (Catalog #400753, #400805, and #400800, respectively). All of Stratagene’s Absolutely RNA kits include RNase-free DNase and offer a rapid on-column DNase treatment protocol for removing contaminating DNA from RNA preparations. cDNA can also be prepared from Stratagene’s QPCR Human Reference Total RNA (Catalog #750500), a high-quality control for quantitative PCR gene-expression analysis. The StrataScript® first strand cDNA synthesis kit (Cat #200420) is recommended for high efficiency cDNA synthesis from total or poly(A)+ RNA. Up to 10% of the cDNA synthesis reaction (up to 2.5 μl per 25 μl PCR reaction) can be amplified with FullVelocity SYBR Green master mix. Primer Design and Concentration For best results, design primers that amplify PCR products 90–300 bp in length. The primers should exhibit a melting temperature (Tm) of approximately 60°C, which is the annealing-extension temperature of the FullVelocity amplification reaction. It is critical to minimize the formation of non-specific amplification products in SYBR Green-based QPCR. This issue becomes more prominent at low target concentrations. To maximize the sensitivity of the assay, 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 earliest Ct and an adequate fluorescence for a given target concentration, with minimal or no formation of primer-dimer. This concentration should be determined empirically. Titrating primers over the range of 50–200 nM (each primer) with a suitable range of template amounts is recommended to identify the optimal primer concentration for each system. Generally, a primer concentration of 75–125 nM (each primer) is satisfactory for most systems. 6 FullVelocity™ SYBR® Green QPCR Master Mix Reference Dye The passive reference dye included in this kit 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. Addition of the reference dye is optional. 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 H2O. If you are using Stratagene’s Mx4000, Mx3000P, or Mx3005P instrument, use the reference dye at a 1:500 dilution, resulting in a final concentration of 30 nM. If you are using the ABI PRISM® 7700 instrument, use the reference dye at a 1:50 dilution, resulting in a final concentration of 300 nM. For instruments that allow excitation at ~584 nm (including most tungsten/halogen lamp-based instruments and instruments equipped with a ~584 nm LED), begin optimization using the reference dye at a final concentration of 30 nM. For instruments that do not allow excitation near 584 nm, (including most laserbased instruments) begin optimization using the reference dye at a final concentration of 300 nM. Magnesium Chloride Concentration The optimal magnesium chloride concentration allows for maximum target amplification with minimal non-specific products and primer-dimers. High levels of Mg2+ tend to favor the formation of spurious products including primer-dimers. The 2× FullVelocity SYBR Green QPCR master mix contains MgCl2 at an optimal concentration of 5 mM, which is suitable for most targets. Further optimization with additional MgCl2 is normally not required. Preparing a Single Reagent Mixture for Multiple Samples If multiple samples containing the same primers are run, Stratagene recommends preparing a single mixture of reaction components and then aliquoting the mixture into individual reaction tubes using a fresh pipet tip for each addition. Preparing a common mixture facilitates the accurate dispensing of reagents, minimizes the loss of reagents during pipetting, and helps to minimize sample-to-sample variation. Mixing and Pipetting Enzyme Solutions The FullVelocity SYBR Green master mix should be mixed gently by inversion without generating bubbles. Bubbles in the final solution may create optical errors during the sample read. * The diluted reference dye, if stored in a light-protected tube at 4°C, can be used within the day for setting up additional assays. FullVelocity™ SYBR® Green QPCR Master Mix 7 Recommended Control Reactions Stratagene recommends performing no-template control (NTC) reactions to identify contamination of reagents or false amplification. NTCs should be run in at least duplicate reactions. 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. dUTP is included instead of dTTP in the FullVelocity SYBR Green QPCR master mix. When dUTP replaces dTTP in PCR amplification, treatment with UNG (Uracil-N-glycosylase, not provided in this kit) can prevent the subsequent reamplification of dU-containing PCR products. UNG acts on single- and double-stranded dU-containing DNA by hydrolysis of uracil-glycosidic bonds at dU-containing DNA sites. When this strategy is used, carry-over contamination is eliminated while template DNA (DNA containing T) is left intact. Fluorescence Detection and Data Acquisition Fluorescence may be detected either in real-time or at the endpoint of cycling using a real-time spectrofluorometric thermal cycler. For endpoint analysis, PCR reactions can be run on any thermal cycler and then analyzed with any fluorescence plate reader that accommodates PCR tubes and detects SYBR Green I dye. If using a fluorescence reader, it is recommended that readings be taken both before and after PCR for comparison. Stratagene’s real-time PCR instruments can be used for this purpose. Assemble the FullVelocity SYBR Green reactions and perform a ‘plate-read’ on the instrument before and after thermocycling. The final ΔCt’s indicate the extent of product formation. Acquisition of real-time data generated by SYBR Green I binding to dsDNA should be performed as recommended by the instrument manufacturer. Data should be collected at the annealing/extension step of the cycling protocol provided in the Protocols section. For the Mx3000P or Mx3005P real-time PCR system, use the dissociation curve included in the default thermal profile for SYBR Green experiments (Segment 3 of the Original Settings default thermal profile). This dissociation curve begins with a 1-minute incubation at 95°C to melt the DNA and then a 30-second incubation at 55°C. This is followed by a ramp up to 95°C with Allpoints data collection performed during the ramp. Data collection during the ramp slows the ramp rate to 0.01°C/sec to allow for a slow melt of the PCR product. Stratagene recommends conducting data analysis with adaptive baseline and moving average ON, and with amplification-based threshold OFF. Under these select conditions, each experiment is analyzed using an adaptive baseline and moving average selected by the Mx3000P or Mx3005P software. 8 FullVelocity™ SYBR® Green QPCR Master Mix PROTOCOLS Preparing the Reactions Notes Once the FullVelocity SYBR Green QPCR master mix is thawed, store the tube on ice while setting up the reactions. Following initial thawing of the master mix, store the unused portion at 4°C. Multiple freeze-thaw cycles should be avoided. It is prudent to set up a no-template control reaction to screen for contamination of reagents or false amplification. Consider performing an endogenous control reaction in a separate tube to distinguish true negative results from PCR inhibition or failure. For information on the use and production of endogenous controls for QPCR, see Reference 3. 1. If the reference dye will be included in the reaction (optional), dilute the dye solution provided to 1:500 (for Stratagene’s Mx3000P, Mx3005P, or Mx4000 instruments) or 1:50 (for the ABI PRISM 7700 instrument) using nuclease-free PCR-grade H2O. See the Reference Dye section of Preprotocol Considerations if using another type of instrument. Note 2. Keep all solutions containing the reference dye protected from light. Combine the components listed below in order. Stratagene recommends preparing a single reagent mixture for replicate experimental reactions and no-template-controls (plus at least one reaction volume excess), using multiples of each component listed below. Reagent Mixture Nuclease-free, PCR-grade H2O to adjust the final volume to 25 μl (including experimental DNA) X μl of upstream primer (optimized concentration) X μl of downstream primer (optimized concentration) 0.375 μl of the diluted reference dye (optional) 12.5 μl of 2× FullVelocity SYBR Green QPCR master mix Note 3. If the concentration of primer has not yet been optimized 75–125 nM is a suitable starting point. Gently mix the reagents without creating bubbles (do not vortex) and then distribute the mixture to the individual PCR reaction tubes. FullVelocity™ SYBR® Green QPCR Master Mix 9 Add X μl of experimental gDNA, cDNA, or plasmid DNA to each experimental reaction. 4. Note 5. High DNA concentrations may inhibit the PCR reaction. Stratagene recommends using 10 ng–10 pg genomic DNA, 5 4x10 to 4 copies of plasmid DNA, or cDNA transcribed from 50 ng-5 pg RNA. Gently mix the reactions without creating bubbles (do not vortex). Note 6. Bubbles interfere with fluorescence detection. Centrifuge the reactions briefly. PCR Cycling Program Place the reactions in a QPCR instrument and run the PCR program below. This protocol has been optimized for 96-well block instruments, such as the Mx3000P, Mx3005P, and Mx4000 instruments, and delivers excellent uniformity. Note The FullVelocity SYBR Green PCR master mix has been optimized specifically to support sensitive and reliable amplification using short denaturation and annealing/extension times. It is highly recommended that researchers use the provided cycling conditions to achieve maximum sensitivity and specificity. Fast Two-Step PCR Program : Plasmid, Genomic DNA, and cDNA Targets Cycles a 10 Duration of cycle Temperature 1 5 minutes 95°C 40 10 seconds 95°C 30 secondsa 60°C Set the temperature cycler to detect and report fluorescence during the annealing/ extension step of each cycle. FullVelocity™ SYBR® Green QPCR Master Mix Dissociation Program Mx3000P® and Mx3005P™ Instruments If using Stratagene’s Mx3000P or Mx3005P instrument, use the default dissociation curve for SYBR Green experiments. This default profile dissociation curve begins with a 1-minute incubation at 95°C to melt the DNA and then a 30-second incubation at 55°C. This is followed by a ramp up to 95°C with Allpoints data collection performed during the ramp. Mx4000® Instrument Incubate the amplified product for 1 minute at 95°C, ramping down to 55°C at a rate of 0.2°C/sec. For the dissociation curve, complete 81 cycles of incubation where the temperature is increased by 0.5°C/cycle, beginning at 55°C and ending at 95°C. Set the duration of each cycle to 30 seconds. Other Instruments If using another instrument, follow the manufacturer’s guidelines for generating dissociation curves. FullVelocity™ SYBR® Green QPCR Master Mix 11 TROUBLESHOOTING Amplification Plot Observation Suggestion Little or no increase in fluorescence with cycling A reagent (primer or template) is missing from the PCR reaction. Set up a new reaction. Ensure the correct dilution of reference dye was used. Optimize the primer concentration. Ensure that the correct amount of template was used and that the template 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 target into an assay that is known to work. Verify that the correct number of cycles was used (40 cycles). Ensure that the primer Tms are compatible with the annealing/extension temperature used (60°C). Analyze the PCR product by gel electrophoresis to determine whether amplification was successful. There is increased fluorescence in control reactions without template One of the reagents (e.g. primers, master mix, water) has been contaminated. Follow the procedures outlined in reference 4 to minimize contamination. Perform decontamination during amplification by including uracil-Nglycosylase (UNG) in the PCR reaction mix. See Preventing Template Cross Contamination in Preprotocol Considerations. The Ct is later than expected when amplifying from high template amounts Inhibition may occur at high template amounts (e.g. >100 ng genomic DNA). Check the DNA template concentration of your sample and verify that the recommended amount was used. Using a higher initial denaturation temperature and longer incubation time (98°C for 7 minutes) may minimize inhibition at high genomic DNA template amounts. Amplification Plot rises and drops at the plateau Some reagent-limiting factor was introduced. Titrate the primer and template to determine whether the amount of either component needs to be increased. Dissociation Plot Observation Suggestion Abundance of primer-dimer and non-specific PCR products Check primer design. Verify that the primers do not hybridize to other target sequences and do not exhibit complementarity at the 3´ ends. Verify that primers exhibit a Tm of approximately 60°C. Redesign primers. Perform a primer titration experiment and determine the concentration that provides the earliest Cts and adequate fluorescence, with minimal primer-dimer formation. Verify that the recommended cycling conditions (10 second denaturation, 30 second annealing/extension) are employed. 12 FullVelocity™ SYBR® Green QPCR Master Mix REFERENCES 1. 2. Molecular Probes, Inc., at http://www.probes.com/media/pis/mp07567.pdf. Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. (1993) Biotechnology (N Y) 11(9):1026-30. Bustin, S. A. (2000) Journal of Molecular Endocrinology 25:169-193. Kwok, S. and Higuchi, R. (1989) Nature 339(6221):237-8. 3. 4. ENDNOTES Absolutely RNA®, Mx3000P®, Mx4000®, and StrataScript® are registered trademarks of Stratagene in the United States. FullVelocity, and Mx3005P are trademarks of Stratagene. ABI PRISM® is a registered trademark of The Perkin-Elmer Corporation. SYBR® is a registered trademark of Molecular Probes, Inc. MSDS INFORMATION The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on Stratagene’s website at http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated MSDS’s in a print-ready format. MSDS documents are not included with product shipments. FullVelocity™ SYBR® Green QPCR Master Mix 13 14 FULLVELOCITY™ SYBR® GREEN QPCR MASTER MIX Catalog #600581, 929581 QUICK-REFERENCE PROTOCOL • If the passive reference dye will be included in the reaction (optional), dilute 1:500 (Mx4000®, Mx3000P®, or Mx3005P™ instruments) or 1:50 (ABI PRISM 7700 instrument). Keep all solutions containing the reference dye protected from light. • Thaw the 2× FullVelocity SYBR Green QPCR master mix and store on ice. Keep the master mix protected from light since it contains SYBR Green I dye. Following initial thawing of the master mix, store the unused portion at 4°C. Note • 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. Nuclease-free, PCR-grade H2O to adjust the final volume to 25 μl X μl of upstream primer (optimized concentration) X μl of downstream primer (optimized concentration) 0.375 μl of diluted reference dye (optional) 12.5 μl of 2× FullVelocity SYBR Green QPCR master mix • Gently mix the components without creating bubbles (do not vortex), then distribute the mixture to the individual PCR reaction tubes. • Add X μl of experimental gDNA, cDNA, or plasmid DNA to each experimental reaction. Note 15 Avoid multiple freeze-thaw cycles. Stratagene recommends using 10 ng–10 pg genomic DNA, 4x105 to 4 copies plasmid DNA, or cDNA transcribed from 50ng–5pg RNA. • Gently mix the reactions without creating bubbles (do not vortex). • Centrifuge the reactions briefly. • Place the reactions in the instrument and run the PCR program below. Note This program is appropriate for most 96-well block instruments, including the Mx3000P, Mx3005P, and Mx4000 instruments. Fast PCR Program Cycles a • Duration of cycle Temperature 1 5 minutes 95°C 40 10seconds 95°C 30 secondsa 60°C Set the temperature cycler to detect and report fluorescence during the annealing/ extension step of each cycle. Run a dissociation curve appropriate to the QPCR instrument. Stratagene’s Mx3000P and Mx3005P instruments: use the default dissociation curve for SYBR Green experiments. See the Protocols section for more information. Stratagene’s Mx4000 instrument: use the following dissociation program. Incubate the amplified product for 1 minute at 95°C, ramping down to 55°C at a rate of 0.2°C/sec. Complete 81 cycles of incubation where the temperature is increased by 0.5°C/cycle, beginning at 55°C and ending at 95°C, with a duration of 30 seconds per cycle. Other instruments: follow the manufacturer’s guidelines for generating dissociation curves. 16