Brilliant Multiplex QPCR Master Mix - Agilent

1 Brilliant Multiplex qpcr Master Mix Instruction Manual Catalog #600553 Revision For Research Use Only. Not for use in diagnostic procedures. 600553-12 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 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 use this product. ORDERING INFORMATION AND TECHNICAL SERVICES Email World Wide Web Telephone Location Telephone United States and Canada 800 227 9770 Austria 01 25125 6800 Benelux 02 404 92 22 Denmark 45 70 13 00 30 Finland 010 802 220 France 0810 446 446 Germany 0800 603 1000 Italy 800 012575 Netherlands 020 547 2600 Spain 901 11 68 90 Sweden 08 506 4 8960 Switzerland 0848 8035 60 UK/Ireland 0845 712 5292 All Other Countries Please visit Brilliant Multiplex qpcr Master Mix CONTENTS Materials Provided.

2 1 Storage Conditions .. 1 Additional Materials Required .. 1 Notices to Purchaser .. 1 Introduction .. 2 Molecular Beacons .. 4 TaqMan Probes .. 4 Endpoint vs. Real-Time 6 Fluorescence Monitoring in Real-Time .. 6 Preprotocol Considerations .. 7 Multiplex PCR .. 7 Magnesium Chloride Concentration .. 8 Preventing Template Cross-Contamination .. 8 Reference Dye .. 9 Fluorescence Detection .. 9 Data Acquisition with a Spectrofluorometric Thermal Cycler .. 9 Protocol: Mx3000P, Mx3005P and Mx4000 Instruments and Other Tungsten Halogen Lamp-Based Platforms .. 10 Troubleshooting: Molecular Beacons .. 12 Troubleshooting: TaqMan Probes .. 13 References .. 14 Endnotes .. 14 MSDS Information.

3 14 Quick-Reference Protocol .. 15 Brilliant Multiplex qpcr Master Mix 1 Brilliant Multiplex qpcr Master Mix MATERIALS PROVIDED Materials provideda Concentration Quantity 2 Multiplex qpcr Master mix 2 ml Reference dyeb 1 mM 100 l a Sufficient PCR reagents are provided for two hundred, 25- l reactions.

4 B The reference dye is light sensitive and should be kept away from light whenever possible. 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 is guaranteed for 6 months. ADDITIONAL MATERIALS REQUIRED Spectrofluorometric thermal cycler Nuclease-free PCR-grade water NOTICES TO PURCHASER NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 6,258,569, 6,171,785, 6,127,155, 6,030,787, 5,994,056, 5,876,930, 5,804,375, 5,789,224, 5,773,258 (claims 1 and 6 only), 5,723,591, 5,677,152 (claims 1 to 23 only), 5,618,711, 5,538,848, and claims outside the US corresponding to expired US Patent No.

5 5,079,352. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

6 Revision Agilent Technologies, Inc. 2015. 2 Brilliant Multiplex qpcr Master Mix INTRODUCTION Quantitative PCR is a powerful tool for gene expression analysis. Many fluorescent chemistries are used to detect and quantitate gene transcripts. The use of fluorescent probe technologies reduces the risk of sample contamination while maintaining convenience, speed, and high throughput screening capabilities. Real-time quantitative PCR can provide both absolute and relative quantification during the exponential phase of amplification. To achieve accurate quantification, the use of a normalizing gene is required. Multiplex qpcr allows one or more targets of interest to be amplified with a normalizing gene in the same reaction vessel by using probes with spectrally different fluorophores for the detection of each amplicon.

7 Unlike conventional qpcr reagents which can amplify up to two targets, Agilent 's Brilliant Multiplex qpcr Master Mix is specifically optimized to amplify up to four targets in one reaction without sacrificing the accuracy of gene transcript quantification. The Brilliant Multiplex qpcr Master mix is provided in a single-tube reagent format, making it ideal for most high-throughput qpcr applications. The Brilliant Multiplex Master mix supports quantitative amplification and detection with Multiplex capability and shows consistent high performance with various fluorescent detection systems, including molecular beacons and TaqMan probes.

8 The Brilliant Multiplex Master mix includes the components necessary to carry out PCR amplifications,* and has been successfully used to amplify and detect a variety of DNA targets, including genomic DNA, plasmid DNA, and cDNA. Including the passive reference dye in a separate tube makes the Brilliant Multiplex Master mix adaptable for many real-time qpcr platforms. * Primers and template are not included. Brilliant Multiplex qpcr Master Mix 3 The Brilliant Multiplex Master mix provides amplification of multiple targets per reaction. The sensitivity is equivalent to that seen in singleplex qpcr reactions, maximizing use of each precious sample without sacrificing sensitivity (see Figure 1).

9 In addition, the Brilliant Multiplex Master mix provides sufficient resources to allow amplification of a low abundance target and a high abundance target in the same tube. The Brilliant Multiplex qpcr Master mix includes SureStart Taq DNA polymerase, a modified version of Taq2000 DNA polymerase with hot start capability. SureStart Taq DNA polymerase improves PCR amplification reactions by decreasing background from non-specific amplification. Using SureStart Taq, hot start is easily incorporated into PCR protocols already optimized with Taq DNA polymerase, with little modification of cycling parameters or reaction conditions. FIGURE 1 Four targets eNOS (FAM), HFE (HEX), CFTR (TR), and Cyclophilin (Cy5) were run in singleplex and Multiplex using the Brilliant Multiplex qpcr Master mix on the Mx3000P real-time PCR system.

10 Ct values for each target were virtually identical in the two reactions, indicating full sensitivity and performance in Multiplex . 4 Brilliant Multiplex qpcr Master Mix Molecular Beacons Molecular beacons are hairpin-shaped fluorescent hybridization probes that can be used to monitor the accumulation of specific product during or after 5 Molecular beacons have a fluorophore and a quencher molecule at opposite ends of an oligonucleotide. The ends of the oligonucleotide are designed to be complementary to each other. When the unhybridized probe is in solution, it adopts a hairpin structure that brings the fluorophore and quencher sufficiently close to each other to allow efficient quenching of the fluorophore.