Precision OneStep TM qrt-pcr Mastermix

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1 Precision OneStep TM qrt-pcr Mastermix Instructions for use of Primerdesign Precision OneStep TM Mastermix for real-time RT-PCR ONE OneStep MasterMix

2 Contents Introduction 3 Kit Contents 5 Recommended Accompanying Products 5 Reagents and Equipment to Be Supplied by User 5 Kit Storage 5 Suitable Sample Material 6 Licensing Agreement and Limitations of Use 6 Primerdesign Ltd Satisfaction Guarantee 6 Quality Control 6 2

3 Introduction 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. Precision OneStep TM qrt-pcr Mastermix contains a unique thermostable MMLV (Moloney murine leukemia virus) enzyme which has an optimal operating temperature of 55C. It also has a higher affinity for primer template duplexes which allows very rapid processing during the RT step. This enables reverse transcription to be completed in under 10 minutes. The Mastermix also contains TAQ Polymerase as well as buffer and MgCl 2 at concentrations optimised for the amplification step. Only the template RNA and primer and probe mix are required to complete the experimental set up for a perfect single tube reaction. Precision OneStep TM qrt-pcr Mastermix has a wide linear range over 8 orders of magnitude and is highly sensitive at detecting low copy number templates that give CT values close to the theoretical limits of detection (Fig.1). Fig 1. Analysis of ACTB expression in a one step qrt-pcr reaction using Precision OneStep qrt-pcr MasterMix. RNA was extracted from HeLa cells and serially diluted (10 x dilutions). 3

4 Guide to Hardware compatibility Manufactures use varying methods to calibrate a real-time PCR reaction. For this reason the correct Precision OneStep TM MasterMix formulation must be use for each platform. Cat Number Product Description Compatible Hardware OneStep Precision OneStep TM qrt-pcr Mastermix MJ Chromo4, Roche lightcycler 480 Platforms OneStep-LR Precision OneStep TM qrt-pcr Mastermix with LOW ROX Applied Biosystems 7500 platform OneStep-R Precision OneStep TM qrt-pcr Mastermix with ROX Applied Biosystems 7700, 7000, 7900, 7300 and ONE STEP platform OneStep-iC OneStep-MX OneStep-CL Precision OneStep TM qrt-pcr Mastermix for the BioRad icycler Precision OneStep TM qrt-pcr Mastermix for Stratagene hardware Precision OneStep TM qrt-pcr Mastermix for capillary lightcyclers BioRad icycler, IQ4 and IQ5 platforms Stratagene MX platforms Roche Capillary Lightcycler platforms If your real-time PCR machine is not listed. Please contact us via our website: SYBRgreen based detection The above kits are for probe based detection chemistry. If you are using primer only kits then the addition of SYBRgreen is required in the Mastermix. Mastermix with SYBRgreen can be supplied at no extra charge by adding "-SY" to catalogue number. 4

5 Kit Contents Precision OneStep TM qrt-pcr Mastermix Includes thermo-stable MMLV and complete qpcr mastermix Precision TM qpcr Mastermix This control reagent lacks the MMLV and is used to make an RT minus control. Recommended Accompanying Products Primerdesign custom designed real-time PCR primers or PrimerDesign pathogen detection kits Primerdesign real-time PCR internal control Primerdesign Bright White TM real-time PCR plasticware Reagents and Equipment to Be Supplied by User Real-Time PCR Instrument Pipettors and Tips Vortex and centrifuge Kit Storage The Primerdesign Precision OneStep TM 2X qpcr Mastermix kit should be stored at -20ºC on arrival. Under these conditions reagents are stable for twelve months from date of purchase. (n.b. Repeated freeze/thawing may compromise the performance of the product). 5

6 Suitable Sample Material All 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 the samples. To prepare a negative-control, replace the test sample with RNAse/DNAse free water. Licensing Agreement and Limitations of Use PCR is covered by several patents owned by Hoffman-Roche Inc and Hoffman-LaRoche, Ltd. Purchase of Primerdesign kits does not include or provide licence with respect to any patents owned by Hoffman-La Roche or others. SYBR green is a registered trademark of Molecular Probes Inc. This product is developed, designed and sold for research purposes only. It is not intended for human diagnostic or drug purposes or to be administered to humans unless clearly expressed for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use. Primerdesign Ltd Satisfaction Guarantee Primerdesign takes pride in the quality of all our products. Should this product fail to perform satisfactorily when used according to the protocols in this manual, Primerdesign will replace the item free of charge. Quality Control As part of our ISO9001 and ISO13485 quality assurance systems, all Primerdesign products are monitored to ensure the highest levels of performance and reliability. 6

7 Bench-side Protocol For optimum performance and sensitivity. All pipetting steps and experimental plate set up should be performed on ice. After the plate is poured proceed immediately to the One Step amplification protocol. Prolonged incubation of reaction mixes at room temperature can lead to PCR artefacts that reduce the sensitivity of detection. When using Primerdesign kits. For each 20µl real-time PCR reaction add the following to each reaction tube Components 1 Reaction Precision OneStep TM qrt-pcr Mastermix * 10 µl Primer/Probe mix 1 µl Template RNA x µl RNAse/DNAse free water x µl Final volume 20 µl * to include an RT minus control substitute Precision TM 2X qpcr Mastermix Suggested use with user supplied primers and probe. For each 20µl real-time PCR reaction add the following to each reaction tube Components 1 Reaction Precision OneStep TM qrt-pcr Mastermix * 10 µl Primers (6pmols Forward and Reverse) x µl Probe (3pmols) x µl Template RNA (25ng) x µl RNAse/DNAse free water x µl Final volume 20 µl *6pmols of primer gives a working concentration of 300nM in a 20µl reaction 7

8 Amplification Protocols For use with Double Dye (Taqman ) gene detection chemistry Cycling x40** Step Time Temp Reverse Transcription 10 min 55ºC Enzyme Activation - HotStart 8 min 95ºC Denaturation 10s 95ºC DATA COLLECTION* 60s 60ºC *Fluorogenic data should be collected during this step through the FAM channel. ** For low copy number targets, giving late detection, a further 10 cycles may be needed to generate the complete amplification plot For use with SYBRgreen detection chemistry Cycling x40*** Step Time Temp Reverse Transcription 10 min 55ºC Enzyme activation - HotStart 8 min 95ºC Denaturation 10s 95ºC DATA COLLECTION* 60s 60ºC Melt Curve** *Fluorogenic data should be collected during this step through the SYBRgreen channel. **A post PCR run melt curve can be used to prove the specificity of the primers. See the manufactures instructions for your hardware platform ** For low copy number targets, giving late detection, a further 10 cycles may be needed to generate the complete amplification plot 8

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