Manual: Brilliant III Ultra-Fast QRT-PCR Master Mix
Brilliant III Ultra-Fast QRT-PCR Master MixInstruction ManualCatalog #600884 (single kit)#600885 (10-pack kit)Revision CResearch Use Only. Not for Use in Diagnostic Procedures.600884-12
LIMITED PRODUCT WARRANTYThis 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 fora 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 usethis product.ORDERING INFORMATION AND TECHNICAL SERVICESUnited States and CanadaAgilent TechnologiesStratagene Products Division11011 North Torrey Pines RoadLa Jolla, CA 92037Telephone(858) 373-6300Order Toll Free(800) 424-5444Technical Services (800) 894-1304emailtechservices@agilent.comWorld Wide Web tria01 25125 6800Benelux02 404 92 22Denmark45 70 13 00 30Finland010 802 220France0810 446 446Germany0800 603 1000Italy800 012575Netherlands020 547 2600Spain901 11 68 90Sweden08 506 4 8960Switzerland0848 8035 60UK/Ireland0845 712 5292All Other CountriesPlease contact your local distributor. A complete list of distributors is available at www.genomics.agilent.com.
Brilliant III Ultra-Fast QRT-PCR Master MixCONTENTSMaterials Provided. 1Storage Conditions . 1Additional Materials Required . 1Notices to Purchaser . 1Introduction. 2Features of Kit Components. 2Fluorescence Monitoring in Real-Time. 3Preprotocol Considerations. 5RNA Isolation. 5Quantitative PCR Human Reference Total RNA . 5Probe Design . 6Optimal Concentrations for Experimental Probes and PCR Primers . 6Preventing Sample Contamination . 6Magnesium Chloride Concentration. 6Reference Dye . 7Reaction Preparation . 7Temperature and Duration of cDNA Synthesis Reaction. 8Multiplex RT-PCR . 8Recommended Control Reactions . 9Protocol . 10Preparing the Reactions. 10RT-PCR Cycling Programs . 11Troubleshooting . 12References . 13Endnotes. 13MSDS Information. 13Quick-Reference Protocol . 15
Brilliant III Ultra-Fast QRT-PCR Master MixMATERIALS PROVIDEDCatalog #600884 (single kit), #600885 (10-pack kit)Materials ProvidedQuantity a,b2 Brilliant III Ultra-Fast QRT-PCR Master Mix2 2 mlRT/RNase Block400 μl100 mM DTT100 μlReference dye , 1 mMcabc100 μlSufficient reagents are provided for four hundred, 20-μl QRT-PCR reactions.Quantities listed are for a single kit. For 10-pack kits, each item is provided at 10 times the listed quantity.The reference dye is light sensitive and should be kept away from light whenever possible.STORAGE CONDITIONSAll Components: Store at –20 C upon receipt. After thawing, the 2 master may be stored at 4 Cfor up to three months or returned to –20 C for long term storageNoteThe reference dye is light sensitive and should be kept away from light whenever possible.ADDITIONAL MATERIALS REQUIREDSpectrofluorometric thermal cyclerNuclease-free PCR-grade waterNOTICES TO PURCHASERUse of labeling reagents may require licenses from entities other than Agilent. For example, use offluorogenic probes in 5' nuclease assays may require licenses under U.S. Patent Nos. 6,214,979,5,804,375, 5,210,015 and 5,487,972 owned by Roche Molecular Systems, Inc. and under U.S. PatentNo. 5,538,848 owned by Applied BiosystemsNotice to Purchaser: Limited LicensePractice of the patented 5’ Nuclease Process requires a license from Applied Biosystems. Thepurchase of this product includes an immunity from suit under patents specified in the product insertto use only the amount purchased for the purchaser's own internal research when used with theseparate purchase of Licensed Probe. No other patent rights are conveyed expressly, by implication,or by estoppel. Further information on purchasing licenses may be obtained from the Director ofLicensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.Revision CBrilliant III Ultra-Fast QRT-PCR Master Mix Agilent Technologies, Inc. 2010.1
INTRODUCTIONQuantitative reverse transcription PCR (QRT-PCR) is a powerful tool forgene expression analysis. The Brilliant III Ultra-Fast QRT-PCR Master Mixwas developed for the ABI StepOnePlus and Bio-Rad CFX96 real-time PCRinstruments and other fast-cycling systems (such as the ABI 7900HT and7500 Fast systems). It performs QRT-PCR in less time withoutcompromising target detection sensitivity, specificity, or reproducibility.The master mix includes two key components that enable it to performoptimally under fast cycling conditions: A mutated form of Taq DNA polymerase that has been specificallyengineered for faster replicationAn improved chemical hot start mechanism that promotes faster hotstart release to improve amplification specificity while keeping therun time of the PCR protocol to a minimumThe master mix has been successfully used with fluorescent TaqMan probes to amplify and detect a variety of high- and low-abundance RNAtargets from experimental samples including total RNA, poly(A) RNA, andsynthetic RNA.The kit includes the components necessary to carry out cDNA synthesis andPCR amplification in one tube and one buffer.* Brilliant kits supportquantitative amplification and detection with multiplex capability. Thesingle-step master mix format is ideal for most high-throughput QPCRapplications where it is not necessary to archive cDNA.Features of Kit ComponentsRT/RNase BlockThe reverse transcriptase (RT) provided in the kit is a Moloney-based RTspecifically formulated for the Stratagene Brilliant III Ultra-Fast kits. ThisRT performs optimally at a reaction temperature of 50 C when used in1-step QRT-PCR with the Brilliant III master mix. It is stringently qualitycontrolled to verify the absence of nuclease contaminants that adverselyaffect cDNA synthesis and to ensure sensitive and reproducible performancein QRT-PCR experiments with a broad range of RNA template amounts anda variety of RNA targets that vary in size, abundance, and GC-content. TheRNase block, provided in the same tube, serves as a safeguard againstcontaminating RNases.Brilliant III Ultra-Fast QRT-PCR 2 Master MixThe 2 master mix contains an optimized RT-PCR buffer, MgCl2,nucleotides (GAUC), stabilizers, and mutant Taq DNA polymerase. TheDNA polymerase features a hot start capability that reduces nonspecificproduct formation.* Primers, probes and template are not included.2Brilliant III Ultra-Fast QRT-PCR Master Mix
DTTA separate tube of 100 mM DTT is provided with the kit. Adding DTT tothe reactions improves RT performance for more challenging targets.Reference dyeA passive reference dye (an optional reaction component) is provided in thekit as an optional reagent that may be added to compensate for non-PCRrelated variations in fluorescence. Providing the reference dye in a separatetube makes the master mix adaptable for many real-time QPCR platforms.Fluorescence Monitoring in Real-TimeWhen fluorescence signal from a PCR reaction is monitored in real-time, theresults can be displayed as an amplification plot, which reflects the changein fluorescence during cycling. This information can be used during PCRexperiments to quantify initial copy number. Studies have shown that initialcopy number can be quantified during real-time PCR analysis based onthreshold cycle (Ct).1 Ct is defined as the cycle at which fluorescence isdetermined to be statistically significant above background. The thresholdcycle is inversely proportional to the log of the initial copy number.1 Themore template that is initially present, the fewer the number of cycles ittakes to reach the point where the fluorescence signal is detectable abovebackground. Quantitative information based on threshold cycle is moreaccurate than information based on endpoint determinations becausethreshold cycle is based on measurements taken during the exponentialphase of PCR amplification when PCR efficiency is not yet influenced bylimiting reagents, small differences in reaction components, or accumulationof PCR inhibitors. Figure 1 shows an ABI StepOnePlus instrumentamplification plot with Ct determination (top panel) and standard curve(bottom panel). In this experiment, the cyclophilin target was amplified anddetected from total RNA using a TaqMan probe and the Brilliant III UltraFast QRT-PCR master mix.Brilliant III Ultra-Fast QRT-PCR Master Mix3
Figure 1 Top panel: StepOnePlus instrument amplification plot using a TaqMan probe. A serial dilution of RNA template(ranging from 0.01–100 ng) was added to each reaction (set up in duplicate) . The fluorescence value used to determine Ct(the threshold line) is shown as a solid line. Bottom panel: Standard curve generated from amplification plot. Anamplification efficiency of 93.2% and an R-squared value of 0.999 were obtained.4Brilliant III Ultra-Fast QRT-PCR Master Mix
PREPROTOCOL CONSIDERATIONSRNA IsolationHigh-quality intact RNA is essential for successful synthesis of full-lengthcDNA. Total and poly(A) RNA can be rapidly isolated and purified usingStratagene Absolutely RNA isolation kits. Oligo(dT)-selection for poly(A) RNA is typically not necessary, although including this step may improvethe yield of specific cDNA templates. RNA samples with OD260/280 ratios of1.8–2.0 are optimally pure.Preventing RNase ContaminationTake precautions to minimize the potential for contamination byribonucleases (RNases). RNA isolation should be performed underRNase-free conditions. Wear gloves and use sterile tubes, pipet tips, andRNase-free water. Do not use DEPC-treated water, which can inhibit PCR.The RNase inhibitor that is included in the tube of RT/RNase Blockprovides additional protection against RNase contamination.Preventing Genomic DNA ContaminationContaminating DNA can be removed from the RNA preparation using anRNase-free DNase. Additionally, PCR primers may be designed to spanadjacent exons in order to prevent amplification of the intron-containinggenomic DNA.Quantitative PCR Human Reference Total RNAStratagene QPCR Human Reference Total RNA (Catalog #750500) is ahigh-quality control for quantitative PCR gene-expression analysis.Stratagene QPCR Human Reference Total RNA is composed of total RNAfrom 10 human cell lines (see the table below), with quantities of RNA fromthe individual cell lines optimized to maximize representation of genetranscripts present in low, medium, and high abundance. The reference RNAis carefully screened for contaminating genomic DNA, the presence ofwhich can complicate interpretation of QRT-PCR assay data.Quantitative PCR Human Reference Total RNA Cell Line DerivationsAdenocarcinoma, mammary glandHepatoblastoma, liverAdenocarcinoma, cervixEmbryonal carcinoma, testisGlioblastoma, brainMelanoma, skinLiposarcomaHistiocytic lymphoma; macrophage; histocyteLymphoblastic leukemia, T lymphoblastPlasmacytoma; myeloma; B lymphocyteBrilliant III Ultra-Fast QRT-PCR Master Mix5
The QPCR Human Reference Total RNA is ideally suited for optimizingQRT-PCR assays. Often only small amounts of experimental RNA templateare available for setting up an expression profiling study. Using theextensive representation of specific mRNA species in the generic template,assays may be optimized for a variety of primer/probe systems. Thiseliminates the use of precious experimental RNA samples for assayoptimization.Probe DesignProbes should have a melting temperature that is 7–10 C higher than theannealing temperature of the primers. For additional considerations indesigning TaqMan probes, refer to Primer Express software from AppliedBiosystems, the Primer3 program or a similar oligo design program.Resuspend lyophilized custom TaqMan probes in buffer containing 5 mMTris-HCl, pH 8.0, and 0.1 mM EDTA (low TE buffer).Optimal Concentrations for Experimental Probes and PCR PrimersProbesThe optimal concentration of the experimental TaqMan probe should bedetermined empirically. The optimal concentration is the lowestconcentration that results in the lowest Ct and an adequate fluorescence for agiven target concentration. The TaqMan probe concentration can beoptimized by varying the final concentration from 100 to 600 nM.PCR PrimersThe optimal concentration of the upstream and downstream PCR primersshould also be determined empirically. The optimal concentration is thelowest concentration that results in the lowest Ct and an adequatefluorescence for a given target concentration. The primer concentration canbe optimized by varying the concentration from 200 to 600 nM. The bestconcentrations of the upstream and downstream primers are not always ofequal molarity.Preventing Sample ContaminationTake precautions to minimize the potential for carryover of nucleic acidsfrom one experiment to the next. Use separate work areas and pipettors forpre- and post-amplification steps. Use positive displacement pipets oraerosol-resistant pipet tips.Treatment with Uracil-N-glycosylase (UNG) is NOT recommended fordecontamination of single tube RT-PCR reactions since UNG would beactive during the 50 C incubation necessary for reverse transcription.Magnesium Chloride ConcentrationMagnesium chloride concentration affects the specificity of the PCR primersand probe hybridization. The Brilliant III QRT-PCR master mix containsMgCl2 at a concentration of 5.5 mM (in the 1 solution), which is suitablefor most targets.6Brilliant III Ultra-Fast QRT-PCR Master Mix
Reference DyeA passive reference dye is included in this kit and may be added tocompensate for non-PCR related variations in fluorescence. Fluorescencefrom the passive reference dye does not change during the course of thePCR reaction but provides a stable baseline to which samples arenormalized. In this way, the reference dye compensates for changes influorescence between wells caused by slight volume differences in reactiontubes. The excitation and emission wavelengths of the reference dye are584 nm and 612 nm, respectively. Although addition of the reference dye isnot required when using the Bio-Rad CFX96 real-time PCR system, withother instruments (including the ABI StepOnePlus instrument) the use of thereference dye may be required for optimal results.Reference Dye Dilution RecommendationsPrepare fresh* dilutions of the reference dye prior to setting up thereactions, and keep all tubes containing the reference dye protected fromlight as much as possible. Make initial dilutions of the reference dye usingnuclease-free PCR-grade H2O. If using a StepOnePlus or 7900HT Fastinstrument, dilute the dye 1:50 for a final concentration of 300 nM in thereactions. For the Stratagene Mx instruments or the ABI 7500 Fastinstrument, dilute the dye 1:500 for a final concentration of 30 nM. The BioRad CFX96, the Roche LightCycler 480 and the QIAGEN Rotor-Gene Qinstruments do not require the use of the reference dye.Reaction PreparationSetting Up Reactions on IceWhile setting up the reactions, keep the reagent mixture and reaction tubeson ice until the reactions are loaded into the instrument.Preparing a Single Mixture for Multiple SamplesIf running multiple samples containing the same primers and probes, preparea single mixture of reaction components and then aliquot the mixture intoindividual reaction tubes using a fresh pipet tip for each addition. Preparinga common mixture facilitates the accurate dispensing of reagents, minimizesthe loss of reagents during pipetting, and helps to minimize sample-tosample variation.Mixing and Pipetting EnzymesSolutions that contain enzymes (including reverse transcriptase and DNApolymerase) should be mixed gently by inversion or gentle vortexingwithout generating bubbles. Pipet the enzymes carefully and slowly;otherwise, the viscosity of the buffer can lead to pipetting errors.* The diluted reference dye, if stored in a light-protected tube at 4 C, can be used within theday for setting up additional assays.Brilliant III Ultra-Fast QRT-PCR Master Mix7
Temperature and Duration of cDNA Synthesis ReactionFor cDNA synthesis, we recommend a 50 C incubation for use with theBrilliant III Ultra-Fast QRT-PCR master mix. A 10-minute incubation forthe first-strand synthesis reaction is sufficient for most targetsMultiplex RT-PCRMultiplex RT-PCR is the amplification of more than one target in a singlepolymerase chain reaction.4 The Brilliant III Ultra-Fast QRT-PCR mastermix has been successfully used to amplify two targets in a multiplexreaction without reoptimizing the concentrations of DNA polymerase,reverse transcriptase or dNTPs.In a typical multiplex RT-PCR reaction, one PCR primer pair primes theamplification of the target of interest and another PCR primer pair primesthe amplification of an endogenous control. For accurate analysis, it isimportant to minimize competition between concurrent amplifications forcommon reagents. To minimize competition, the limiting primerconcentrations need to be determined.5 Consideration should also be givento optimization of the other reaction components. The number offluorophores in each tube can influence the analysis. T
2 Brilliant III Ultra-Fast QRT-PCR Master Mix INTRODUCTION Quantitative reverse transcription PCR (QRT-PCR) is a powerful tool for gene expression analysis. The Brilliant III Ultra-Fast QRT-PCR Master Mix was developed for the ABI StepOnePlus and Bio-Rad CFX96 real-time PCR instruments and other fast-cycling systems (such as the ABI 7900HT and
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