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Precision PLUSOneStepRT-qPCR Master MixInstructions for use of PrimerdesignPrecision PLUS OneStep Master Mix for real-time RT-qPCR
ContentsIntroduction3Kit contents5Recommended accompanying products5Reagents and equipment to be supplied by user5Kit storage6Suitable sample material5Licensing agreement and limitations of use6Primerdesign Ltd satisfaction guarantee6Quality control6Precision PLUS OneStep RT-qPCR Master Mix 1mlHandbook HB04.06.03Published Date: 07/12/20182
IntroductionPrecisionPLUS OneStep RT-qPCR Master Mix is an optimised complete system foruse in one step real-time PCR. Removal of a separate reverse transcription stepreduces handling errors and greatly reduces the time taken to obtain results.PrecisionPLUS OneStep RT-qPCR Master Mix contains a unique thermostable MMLV(Moloney murine leukemia virus) enzyme which has an optimal operating temperatureof 55 C. It also has a higher affinity for primer template duplexes which allows veryrapid processing during the RT step. This enables reverse transcription to becompleted in under 10 minutes. The Master Mix also contains Taq Polymerase as wellas buffer and MgCl2 at concentrations optimised for the amplification step. Only theRNA template and primer and probe mix are required to complete the experimental setup for a perfect single tube reaction.Precision PLUS OneStep RT-qPCR Master Mix 1mlHandbook HB04.06.03Published Date: 07/12/20183
Guide to Hardware compatibilityManufacturers use varying methods to calibrate a real-time PCR reaction. For thisreason, the correct PrecisionPLUS OneStep Master Mix formulation must be used foreach platform.Cat NumberProduct DescriptionCompatible HardwareMJ Opticon and Chromo4Roche lightcycler 480 and LC96Biorad CFXRotorgene, EppendorfMastercyclerFluidigm BioMarkPrecision PLUS OneStep RT-qPCR Master MixCepheid SmartCyclerIllumina EcoPCRMax EcoThermo PikoRealPrimerPro 48Analytik Jena qTower SeriesApplied Biosystems 7500Precision PLUS OneStep RT-qPCR Master MixViiA7with LOW ROXLife Technologies QuantStudioOSPLUSOSPLUS-LROSPLUS-RApplied Biosystems 7000Applied Biosystems 7300Applied Biosystems 7700 Precision PLUS OneStep RT-qPCR Master MixApplied Biosystems 7900with ROXApplied Biosystems StepOneAppliedBiosystems StepOnePLUSGeneAmp 5700 platformsOSPLUS-ICPrecision PLUS OneStep RT-qPCR Master MixBioRad iCycler IQ4 and IQ5for the BioRad iCyclerOSPLUS-MXPrecision PLUS OneStep RT-qPCR Master MixStratagene MX platformsfor Stratagene hardwareOSPLUS-CLPrecision PLUS OneStep RT-qPCR Master Mix Roche Capillary Lightcycler 1.5for capillary lightcyclersand 2.0.If your real-time PCR machine is not listed.www.primerdesign.co.ukPlease contact us via our website:Pack sizesThe following pack sizes are available:Cat NumberPack size CompositionOSPLUS-1ML1ml1 X 1ml aliquotOSPLUS-2ML2ml2 X 1ml aliquotsOSPLUS-5ML5ml5 X 1ml aliquotsOSPLUS-10ML10ml10 X 1ml aliquotsOSPLUS-20ML20ml20 X 1ml aliquotsJust add the required pack size to the catalogue numberPrecision PLUS OneStep RT-qPCR Master Mix 1mlHandbook HB04.06.03Published Date: 07/12/20184
SYBR green based detectionIf SYBR green is required in the mix then add ‘–SY’ to the catalogue number e.g.OSPLUS-R-SYInert blue loading dyeIf inert blue loading dye is required, then choose the relevant catalogue number belowand add this to your order when purchasing PrecisionPLUS OneStep Master Mix. Youwill be supplied with a separate vial of blue dye.To use the inert blue loading dye, simply add 4.7µl to each 1ml vial of PrecisionPLUSOneStep Master Mix. Mix the blue dye into the Master Mix by inverting the tube.Cat NumberCompositionBLUEDYE-1MLBlue dye required to convert 1ml Master MixBLUEDYE-2MLBlue dye required to convert 2ml Master MixBLUEDYE-5MLBlue dye required to convert 5ml Master MixBLUEDYE-10MLBlue dye required to convert 10ml Master MixBLUEDYE-20MLBlue dye required to convert 20ml Master MixKit contents PrecisionPLUS OneStep RT-qPCR Master Mix aliquotsRecommended accompanying products Primerdesign custom designed real-time PCR primers genesig pathogen detection kits Primerdesign real-time PCR internal control Primerdesign BrightWhite real-time PCR plasticwareReagents and equipment to be supplied by user Real-Time PCR Instrument Pipettes and Tips Vortex and centrifugePrecision PLUS OneStep RT-qPCR Master Mix 1mlHandbook HB04.06.03Published Date: 07/12/20185
Kit storageThe Primerdesign PrecisionPLUS OneStep RT-qPCR Master Mix kit should be storedat -20ºC on arrival. Repeated freeze/thawing should be avoided as it may compromisethe performance of the product. Primerdesign does not recommend using the kit afterthe expiry date stated on the pack.Suitable sample materialAll kinds of RNA sample material can be used (e.g. Viral RNA, cell culture derived RNA,Biopsy derived RNA etc). Please ensure the samples are suitable in terms of purity,concentration and RNA integrity. Always run at least one negative control with thesamples. To prepare a negative control, replace the test sample with RNase/DNasefree water.Licensing agreement and limitations of usePCR is covered by several patents owned by Hoffman-Roche Inc and HoffmanLaRoche Ltd. Purchase of Primerdesign kits does not include or provide licence withrespect to any patents owned by Hoffman-La Roche or others. SYBR green is aregistered trademark of Molecular Probes Inc. PrecisionPLUS containing GoTaq HotStart Polymerase manufactured by Promega Corporation for distribution byPrimerdesign Ltd. Licensed to Promega under U.S. Patent Nos. 5,338,671 and5,587,287 and their corresponding foreign patents.Primerdesign Ltd satisfaction guaranteePrimerdesign takes pride in the quality of all our products. Should this product fail toperform satisfactorily when used according to the protocols in this manual,Primerdesign will replace the item free of charge.Quality controlAs part of our ISO9001 and ISO13485 quality assurance systems, all Primerdesignproducts are monitored to ensure the highest levels of performance and reliability.Precision PLUS OneStep RT-qPCR Master Mix 1mlHandbook HB04.06.03Published Date: 07/12/20186
Bench-side protocolFor optimum performance and sensitivity all pipetting steps and experimental plate set upshould be performed on ice. After the plate is poured proceed immediately to the onestep amplification protocol. Prolonged incubation of reaction mixes at room temperaturecan lead to PCR artefacts that reduce the sensitivity of detection.When using Primerdesign kits:For each 20µl one-step real-time PCR reaction add the following to each reaction tubeComponentsPrecisionPLUS OneStep RT-qPCR Master MixPrimer/Probe mixTemplate RNA (25ng)RNase/DNase free water (up to Final volume)Final volume1 Reaction10 µl1 µlx µlx µl20 µlSuggested use with user supplied primers and probe:For each 20µl one-step real-time PCR reaction add the following to each reaction tubeComponentsPrecisionPLUS OneStep RT-qPCR Master MixPrimers* (6pmols forward and reverse)Probe (3pmols)Template RNA (25ng)RNase/DNase free waterFinal volume1 Reaction10 µlx µlx µlx µlx µl20 µl* 6pmols of primer gives a working concentration of 300nM in a 20µl reactionPrecision PLUS OneStep RT-qPCR Master Mix 1mlHandbook HB04.06.03Published Date: 07/12/20187
Amplification protocolsPrecision PLUS OneStep RT-qPCR Master MixFor use with standard cycling protocols with double-dye gene detection kitsStepReverse TranscriptionEnzyme ActivationDenaturationDATA COLLECTION*Cycling x40**Time10 min2 min10 sec60 secTemp55ºC95ºC95ºC60ºC*Fluorogenic data should be collected during this step through the appropriate channel.** For low copy number targets, giving late detection, a further 10 cycles may be needed to generate thecomplete amplification plotFor use with SYBR green detection chemistryCycling x40***StepReverse TranscriptionEnzyme activationDenaturationDATA COLLECTION*Melt Curve**Time10 min2 min10 sec60 secTemp55ºC95ºC95ºC60ºC*Fluorogenic data should be collected during this step through the SYBR green channel.**A post PCR run melt curve can be used to prove the specificity of the primers. See the manufacturesinstructions for your hardware platform*** For low copy number targets, giving late detection, a further 10 cycles may be needed to generatethe complete amplification plot.Precision PLUS OneStep RT-qPCR Master Mix 1mlHandbook HB04.06.03Published Date: 07/12/20188
Precision PLUS OneStep RT-qPCR Master Mix 1ml Handbook HB04.06.03 Published Date: 07/12/2018 Introduction PrecisionPLUS OneStep RT-qPCR Master Mix is an optimised complete system for use in one step real-time PCR. Removal of a separate reverse transcription step reduces handling errors and greatly reduces the time taken to obtain results.
