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User`s Manual and Instructions

1. 49 0 69 779099 Fax 44 0 1235 820 482 Fax 41 0 91 605 17 85 Fax 49 0 69 13376880 23591 El Toro Rd Suite 167 Lake Forest CA 92630 Tel 1 800 987 0985 Fax 1 949 265 7703 amsbio Eva QPCR SuperMix Kit 101506V2 See page 5 Use of the ROX Reference Dye Final template concentration varies depending on the copy number of target present in the template solution Optimal amount should be determined by preparing the dilution series It is recommended to use DNA template in less than 100 ng 5 Gently mix the reactions without creating bubbles since bubbles interfere with fluorescence detection Then centrifuge the reactions briefly 6 Place the reactions in the instrument and run the appropriate PCR program Try the following protocol firstly and optimize the reaction condition if needed PCR program for amplification Cycles Temperature Time Detection Remark 1 95 C 7 10 min OFF This step will activate the Taq polymerase 40 95 C 30 sec OFF Set the instrument to detect 55 65 C 1 min ON and report fluorescence either 72 30 sec to 1 5 min OFF at the annealing step or the extension step of each cycle 1 72 C 3 min OFF This step can be omitted if the amplicon size is lt 300 bp a Set an appropriate annealing temperature for the primer set used b Set the extension time to 1 1 5 min if the amplicon size is gt 400 bp 2 Disso
2. Eva QPCR SuperMix Kit User s Manual and Instructions Product Eva QPCR SuperMix Kit Catalog Number K5052200 K5052400 Introduction Real time or quantitative PCR QPCR allows quantification of DNA cDNA or RNA templates QPCR is based on the detection of a fluorescent reporter molecule that increases as PCR products accumulate with each cycle of amplification In BioChain s Eva QPCR SuperMix a superior green fluorescence DNA binding dye is used for real time detection and quantitation of DNA The Eva QPCR SuperMix is a ready to use 2x concentrated master mix that contains all the reagents except primers and templates needed for running quantitative real time DNA detection assays in the double stranded DNA dye detection format The passive reference dye ROX is included in a separate tube to make the Eva Supermix adaptable for many real time QPCR platforms A pair of human beta actin primers is also included in the kit as a control BioChain s QPCR SuperMix contains BioChain s Taq polymerase with hot start capability BioChain s hot start Taq polymerase improves PCR amplification reactions by decreasing non specific amplification and preventing primer dimer formation This enzyme is activated after an initial seven to ten minutes heating at 95 C And the real time PCR buffer is specially formulated to provide superior specificity and increase amplification efficiency This SuperMix can amplify and detect a broad range of DNA
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4. or cDNA targets including those are GC or AT rich Eva Dye Eva Dye binds double stranded DNA Detection is monitored by measuring the increase in fluorescence intensity throughout the cycle Eva Dye has higher affinity to double stranded DNA than SYBR Green dye and shows stronger fluorescence intensity than SYBR Green upon binding to DNA Eva Dye is more stable than SYBR Green and the absorption and emission spectra of Eva Dye are similar to SYBR Green Dye or FAM so the same optical setting for SYBR Green Dye or FAM can also be used for Eva Dye UK amp Rest of World Switzerland Deutschland United States Centro Nord Sud 2E CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 Bockenheimer Landstr 17 19 60325 Frankfurt Main Tel 49 0 69 779099 Fax 49 0 69 13376880 23591 El Toro Rd Suite 167 Lake Forest CA 92630 Tel 1 800 987 0985 Fax 1 949 265 7703 184 Milton Park Abingdon OX14 4SE Oxon UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 amsbio Eva QPCR SuperMix Kit 101506V2 Fluorescence dRn 2 4 6 8 10 12 4 16 18 20 22 24 26 28 ko 32 4 3 38 40 Cycles Y 3 289Lo0g x 37 56 R 0 999 Efficiency 101 4 Ot dRn 1x10 1x10 1x10 1x10 1x10 1x10 1x108 Initial Quantity Copy Number Figure 1 BioChain Eva QPCR SuperMix amplifies over a broad dynamic range 2 x 101 to 2 x 108 copies of plasmid containing cDN
5. 591 El Toro Rd Suite 167 p ail C amspio com Lake Forest CA 92630 inic Tel 1 800 987 0985 Fax 1 949 265 7703 Eva QPCR SuperMix Kit 101506V2 1 95 C 10 min OFF 40 95 C 30 sec OFF 55 65 C 1 min ON 72 C 30 sec OFF 3 Dissociation Program Follow manufacturer s guidelines for setting up dissociation depending on the instrument s software version Related Products Pro QPCR SuperMix Cat K5053200 K5053400 dNTP set for PCR Cat K6011100 PCR mix Cat 5051100 PCR Optimization Kit K5051100 Taq Polymerase Cat 7051200 RNA PCR ready cDNA and PCR ready genomic DNA References 1 Biotium Inc at http www biotium com product product_info allcolor pdf 2 Higuchi R Dollinger G Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences BioTechnology 10 413 417 3 Higuchi R Fockler C Dollinger G and Watson R 1993 Kinetic PCR analysis real time monitoring of DNA amplification reactions BioTechnology 11 1026 1030 Deutschland UK amp Rest of World Switzerland United States a m sbi re Bockenheimer Landstr 17 19 23591 El Toro Rd Suite 167 60325 Frankfurt Main Lake Forest CA 92630 Tel 49 0 69 779099 Tel 1 800 987 0985 Fax 49 0 69 13376880 Fax 1 949 265 7703 Centro Nord Sud 2E CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton
6. A of human beta actin gene were amplified in 25 ul reactions Highly reproducible triplicates demonstrated good linearity of 0 999 and excellent PCR efficiency of 101 4 over an 8 order of dynamic range BioChain s Eva SuperMix has high sensitivity detecting as few as 20 copies of target DNA within the linear range Fluorescence R T Temperature C Figure 2 Dissociation Curve of PCR products amplifies over a broad dynamic range 2 x 7 0 to2x 10 copies of plasmid containing cDNA of human f actin gene were amplified in 25 ul reactions ea UK amp Rest of World Switzerland 184 Milton Park Abingdon Centro Nord Sud 2E OX14 4SE Oxon UK CH 6934 Bioggio Lugano Tel 44 0 1235 828 200 Tel 41 0 91 604 55 22 Fax 44 0 1235 820 482 Fax 41 0 91 605 17 85 Deutschland United States Bockenheimer Landstr 17 19 60325 Frankfurt Main Lake Forest CA 92630 Tel 49 0 69 779099 Tel 1 800 987 0985 Fax 49 0 69 13376880 Fax 1 949 265 7703 23591 El Toro Rd Suite 167 Eva QPCR SuperMix Kit 101506V2 Features e Convenient All reaction components are supplied for quick and easy set up e Save time Ready to use SuperMix reduces setup time and liquid handling steps e Wide dynamic range good linearity and excellent PCR efficiency over an 8 orders of dynamic range e High Sensitivity detect as low as 20 copies of DNA e Amplify and detect a broad range of DNA or cDNA tar
7. C for ready use Avoid direct light in preparation of the PCR reaction mixture because Eva Dye is light sensitive 1 If the ROX reference dye will be included in the reaction keep all solutions containing the ROX protected from light 2 Optional Set up a no template control to screen for contamination of reagents or false amplification 3 Due to the sensitivity of quantitative PCR results can be easily affected by pipetting errors Always prepare a master mix of Eva SuperMix containing the primers and the reference dye if reference dye is used Individual pipetting of replicate samples is not recommended Real time PCR Cycling Programs 4 Prepare the following PCR reaction mixture First make the master mix without the template After making the master mix gently mix the reaction without creating the bubbles aliquot and then add 2 ul of template to each experimental reaction per reaction 25 ul Regents Volume Final Concentration Eva QPCR SuperMix 2x 12 5 ul 1x PCR forward primer X pl 100 200 nM PCR reverse primer X ul 100 200 nM Reference Dye ROX 0 5 ul Template 2 ul Nuclease free PCR grade water Add up to 25 ul UK amp Rest of World Switzerland Deutschland United States 184 Milton Park Abingdon Centro Nord Sud 2E Bockenheimer Landstr 17 19 OX14 4SE Oxon UK CH 6934 Bioggio Lugano 60325 Frankfurt Main Tel 44 0 1235 828 200 Tel 41 0 91 604 55 22 Tel
8. Tel 49 0 69 779099 Tel 1 800 987 0985 Fax 44 0 1235 820 482 Fax 41 0 91 605 17 85 Fax 49 0 69 13376880 Fax 1 949 265 7703 Eva QPCR SuperMix Kit 101506V2 Protocol Using Stratagene s Mx3000P Mx4000 and ABI PRISM GENEAmp 5700 Real time PCR Instrument Use of the ROX Reference Dye ROX reference dye is included in this kit and may be added to compensate for non PCR related variations in fluorescence Addition of the reference dye is optional Optimizing the ROX dye concentration within the qPCR reaction is an important aspect of setup Too much ROX in the qPCR reaction will reduce background but also makes a low target signal difficult to distinguish from background Conversely too little ROX can increase background meaning that low or weak target signals can be lost For instruments that allow excitation at 584 nm such as Stratagene s Mx instrument and ABI 7500 firstly 1 10 dilute the ROX reference dye provided in the kit then begin optimization using 0 5 ul diluted ROX reference dye in 25 ul RT PCR reaction For instruments that do not allow excitation near 584 nm such as ABI PRISM GENEAmp 5700 instrument begin optimization using 0 5 ul undiluted ROX reference dye in 25 ul qRT PCR reaction Reagent Preparation and Storage Thaw the tube containing Eva QPCR SuperMix on ice and store it on ice while setting up the reactions After initial thawing aliquot the supermix and store a portion at 4
9. ciation Program for all PCR products Follow manufacturer s guidelines for setting up dissociation depending on the instrument s software version PCR Setup and Cycling Program for human f actin control primer set amplicon size 202 bp 1 Prepare the following PCR reaction mixture First make the master mix without the template After making the master mix gently mix the reaction without creating the bubbles aliquot and then add 2 ul of template to each experimental reaction er reaction 25 ul Regents Volume Eva QPCR SuperMix 2x 12 5 ul Human B actin primer set 25x 1 ul Reference Dye ROX 0 5 ul Template 2 ul Nuclease free PCR grade water Add up to 25 ul See page 5 Use of the ROX Reference Dye Final template concentration varies depending on the copy number of target present in the template solution Optimal amount should be determined by preparing the dilution series 2 PCR program for amplification of human B actin amplicon Cycles Temperature Time Detection United States UK amp Rest of World Switzerland Deutschland 184 Milton Park Abingdon Centro Nord Sud 2E Bockenheimer Landstr 17 19 OX14 4SE Oxon UK CH 6934 Bioggio Lugano 60325 Frankfurt Main Tel 44 0 1235 828 200 Tel 41 0 91 604 55 22 Tel 49 0 69 779099 Fax 44 0 1235 820 482 Fax 41 0 91 605 17 85 Fax 49 0 69 13376880 amsbio 23
10. gets including those that are GC or AT rich e Flexible Compatible with most of the real time PCR instruments Applications e Real Time PCR e Gene expression profiling e Gene knockdown verification e Array validation Description Components in this kit are prepared with pure chemicals according to our proprietary technology BioChain s QPCR SuperMix is a 2x concentration of premix reagent including Hotstart DNA polymerase and Eva Dye and specially formulated real time buffer designed for real time PCR with intercalator format Quality Control 1 kit of this lot has been tested for amplifying plasmid containing human B actin cDNA amplified fragment 202 bp over an 8 orders of dynamic range using Stratagene s Mx3005P as a real time PCR instrument Good linearity and great PCR efficiency is observed and consistent with the previous lot Components Eva QPCR SuperMix Kit Catalog Number K5052200 Reagents are sufficient for 200 assays Item Amount Part No 1 Eva QPCR SuperMix 1 25 ml x 2 K5052200 1 2 ROX reference Dye 50 pl x2 K5052200 2 3 human B actin control F R primer pair 25x 50 pl K5052200 3 Catalog Number K5052400 Reagents are sufficient for 400 assays Item Amount Part No 1 Eva QPCR SuperMix 1 25 ml x 4 K5052400 1 2 ROX reference Dye 50 ul x 4 K5052400 2 3 human B actin control F R primer pair 25x 50 pl K5052400 3 3 UK amp Rest of W
11. orld Switzerland Deutschland United States 184 Milton Park Abingdon Centro Nord Sud 2E Bockenheimer Landstr 17 19 OX14 4SE Oxon UK CH 6934 Bioggio Lugano 60325 Frankfurt Main Tel 44 0 1235 828 200 Tel 41 0 91 604 55 22 Tel 49 0 69 779099 Fax 44 0 1235 820 482 Fax 41 0 91 605 17 85 Fax 49 0 69 13376880 23591 El Toro Rd Suite 167 Lake Forest CA 92630 Tel 1 800 987 0985 Fax 1 949 265 7703 amsbio amspl Eva QPCR SuperMix Kit 101506V2 Reagents and Equipments Required but not Supplied in this Kit 1 Nuclease free PCR grade water 2 Spectrofluorometric thermal cycler Storage and Stability Upon receipt store all components at 20 C in a constant temperature freezer Avoid repeated freeze thaw cycles When stored under these conditions the supermix is stable for one year after ship date You may aliquot the supermix and store a portion at 4 C for ready use The thawed Eva QPCR SuperMix is stable at least for 3 months at 4 C The Eva Dye and the ROX reference dye are light sensitive and should be kept away from light whenever possible UK amp Rest of World Switzerland Deutschland United States 184 Milton Park Abingdon Centro Nord Sud 2E Bockenheimer Landstr 17 19 23591 El Toro Rd Suite 167 OX14 4SE Oxon UK CH 6934 Bioggio Lugano 60325 Frankfurt Main Lake Forest CA 92630 Tel 44 0 1235 828 200 Tel 41 0 91 604 55 22

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