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1、StepOne and StepOnePlus Real-Time PCR Systems Remarkably Simple Systems. Simply Remarkable Results. PRODUCT BROCHURE Real-Time PCR Systems 2 Step Up To High-Performance Real-Time PCR Remarkably Simple Systems Applied Biosystems, a leader in technology solutions for life science research, introduces
2、its latest innovations in real-time PCRthe StepOne (48-well) and StepOnePlus (96-well) Real-Time PCR Systems. These remarkably simple real-time PCR systems are specifically designed with a user-friendly, yet powerful interface for both new and experienced real-time PCR researchers. 3 System Features
3、 The StepOne and StepOnePlus Systems bring advanced real-time PCR technology to a new level of accessibilityeven for first-time users. Both systems measure amplification as it occurs, cycle by cycle, allowing for precise and quantitative measurements during the exponential phase of PCR. Beginning at
4、 the homepage (Figure 1), you can navigate seamlessly through all aspects of the real-time PCR process including sample and reaction setup, thermal cycling, and fluorescent detection. Focused application software analyzes and interprets experimental results. Depending on the experimental design, the
5、 system can even help you select and order real-time PCR reagents online by means of convenient links in the software (optional). The system provides 2-fold discrimination with 99.7% confidence, along with 9 logs of dynamic range. Highly sensitive, these systems detect 10 copies of RNaseP in a 30-L
6、reaction. Additionally, for laboratories with the StepOne System that require greater throughput, an upgrade kit is available.* StepOnePlus Real-Time PCR System Upgrade Kit To accommodate your changing real-time PCR needs, Applied Biosystems offers the StepOnePlus Real-Time PCR System Upgrade Kit. T
7、he upgrade converts a StepOne System into a StepOnePlus System by upgrading the 48-well block to a 96-well VeriFlex Block and expanding the dye flexibility to four colors. The color of the bar above the block is also updated to indicate that the instrument is now a StepOnePlus System. Just send in y
8、our existing StepOne System and youll receive a StepOnePlus loaner instrument to use until your upgraded system arrives in your lab. Easy-to-Use Experimental Design Wizard Both systems provide an experimental Design Wizard to assist you in setting up your first real-time PCR experiment. Just click o
9、n the Design Wizard and start answering questions about the real- time PCR experiment you wish to perform. The Wizard walks you through quantitation methods, detection chemistry, ramp speeds, and nucleic acid template types. From standards to plate layout, the Design Wizard guides the entire process
10、, including reaction mix and standard dilution calculations. Youre now ready to explore the simply remarkable features of these systems. Figure 1. Software Homepage. StepOne System StepOnePlus System Throughput/Wells 48 96 FAM/SYBR Green dyes VIC/JOE dyes ROX dye NED/TAMRA dyes VeriFlex Block Featur
11、es At A Glance System Highlights For both novice and experienced researchers, the StepOne or StepOnePlus Systems have what you need. Cost-effective, 3-color/48-well (StepOne) or 4-color/96-well (StepOnePlus) Systems deliver precise, quantitative real-time PCR results Long-life LED-based optical syst
12、em records fluorescence from FAM/SYBR Green I, VIC/JOE, and ROX dyes for gene expression analysis, pathogen quantitation, SNP genotyping, and presence/absence assays. Note: The StepOnePlus System also accommodates the TAMRA dye Both systems perform Fast PCR reactions in less than 40 minutes Ultra-co
13、mpact footprint fits any laboratory setting LCD touchscreen and USB drive provide configuration flexibility and enable PC free operation Remote monitoring and email notification for convenience and time-savings The StepOnePlus System features VeriFlex Block technology, which combines six independent
14、ly controllable peltier blocks for enhanced PCR functionality and precise temperature control The StepOne System is upgradeable to the StepOnePlus System to meet your changing research needs *StepOnePlus Real-Time PCR System Upgrade Kit 3 4 Flexibility Both the StepOne and StepOnePlus Systems adapt
15、to almost any workflow with flexible instrument control and data management. From the touchscreen control panel, you can quick-start your experimental run without PC connectivity. You can also create a new protocol, view the history of your last run, or see protocol details (Figures 2 and 3). When t
16、he run is complete, data can be downloaded easily onto a USB flash stick or saved to a PC. Either system can be connected directly to a Local Area Network (LAN), and you can monitor the progress of the experiment, send new instructions to the system, download data, and edit the instru- ment profile.
17、 The software for the two systems also contains a convenient email feature that notifies you when your experiment is complete and ready for analysis. The StepOne and StepOnePlus Systems can be installed in multiple distinct configurations, providing unmatched flexibility and convenience. The unique
18、standalone (PC free) configuration provides an ultra-compact footprint that will fit into any laboratory. A direct connection to a LAN enables remote monitoring of experimental progress and downloading of the completed run file to the PC at your desk. System Configurations (Figure 4) 1. PC controlle
19、d 2. PC free 3. Networked 4. PC controlled connected to LAN 5. PC controlled with networked instrument Simply Remarkable Interface 5. PC controlled with network instrument LAN 1. PC controlled4. PC controlled connected to LAN USB10/100 Adapter 2. PC free3. Networked LAN Figure 4. Five Distinct Confi
20、gurations. Figure 2. Browse Experiment Page On Touchscreen.Figure 3. Run Monitor Screen On Touchscreen. 5 Enter known efficiencies to adjust RQ values for each target View amplification plot, multicomponent plot, and QCsummary within the study to easily identify and eliminate outliers Preview the ef
21、fect of modified analysis settings before permanently applying them to results Figure 5. StepOne System Multiple Plots View. The StepOne Software v2.1 includes the Gene Expression Study Package to analyze an unlimited number of plates in one study and displays the results in a publication-ready fash
22、ion. Figure 6. StepOne System With the Gene Expression Study Package. Software The StepOne and StepOnePlus Systems software contains unique features not available in other real-time PCR instruments. For example, data can be analyzed from multiple perspectives in the Multiple Plots view (Figure 5). T
23、he software constructs four-plot, side-by-side views of all data aspects including the amplification plot, standard curve, multi-component data plots, and raw data. It also displays this data next to the plate layout for easier analysis. Another novel feature is the softwares ability to automaticall
24、y identify wells that might compromise the success of an experiment. During data analysis, the software generates a quality-control report table that flags wells based on criteria such as amplification in a negative-control well, the absence of a signal in a well, or a high CT standard deviation in
25、a replicate group. This feature reduces analysis time and allows even researchers new to real-time PCR to have confidence in their results. Furthermore, you can customize this feature by disabling or modifying quality flag settings to suit your experimental needs. Software Highlights Experimental De
26、sign Wizards to help you design and set up experiments Pipetting protocols and recipes to set up experiments quickly Advanced setup if you require more flexibility for complex applications such as multiplexing Quick-start setup so you can begin a run immediately and enter plate information later Rea
27、l-time monitoring of amplification growth curves enables you to view run progress (can be viewed from a remote PC) Multiple Plots view for simultaneous data assessment from four perspectives Remote monitoring and email notification with run file attached for immediate access to resultseven remotely
28、Export easily to PowerPoint , Excel, or directly as a .jpeg file Powerful Gene Expression Study Package (Figure 6) Import an unlimited number of comparative CT (relative quantitation) plates to one study View data by biological replicate group or technical replicate group Normalize to multiple endog
29、enous controls 5. PC controlled with network instrument LAN 1. PC controlled4. PC controlled connected to LAN USB10/100 Adapter 2. PC free3. Networked LAN 6 Applications The StepOne Systems support any real-time PCR application. Predesigned or custom assays exist for the following applications: SNP
30、Genotyping Translocation Analysis Gene Expression Profiling Gene Detection MicroRNA Expression Viral Load Analysis For information about existing Gene Expression, MicroRNA, and Translocation Analysis Assays, please visit . SNP Genotyping Assay information can be found at . Speed The StepOne and Step
31、OnePlus Systems perform both standard and Fast thermal cycling on the same block with no modification. Standard thermal cycling requires less than two hours, and Fast thermal cycling significantly reduces the run times of quantitative real-time PCR applications by delivering results in 40 minutes. F
32、ast cycling is ideal for maximizing the number of runs on an instrument in any given workday. 3.1 2.1 1.1 0.1 0.00.20.40.60.81.01.21.41.61.82.0 Allele 2 Allele 1 Allele Discrimination Plot (SNP Assay: SNP Assay 1) Homozygous 2/2UndetermindedHomozygous 1/1Heterozygous 1/2 4.1 3.6 2.6 0.6 1.6 StepOne
33、and StepOnePlus software automatically determines genotypes and gen- erates an intuitive graphic representation of results in a cluster plot report that helps you view data across populations or samples. Results are from human CYP2C19*2 TaqMan SNP Genotyping Assay (using the StepOnePlus System). Fig
34、ure 8. Genotyping Analysis.Figure 7. Broad Dynamic Range. 0.1 1 10 Rn 246810121416182022242628303234363840 Amplifi cation Plot Cycle Amplification plot shows log of change in normalized reporter fluorescence plot- ted vs. PCR cycle number. This plot from the StepOne System illustrates 9 logs of line
35、ar dynamic range for a TaqMan Assay of cDNA containing the 18S target sequence in ten-fold serial dilutions. Simply Remarkable Applications Instrument Chemistries Both StepOne Systems are equipped with fluorophore detection chemistries that include FAM and VIC dye-labeled TaqMan MGB probe-based assa
36、ys, VIC and TAMRA dye-labeled probe-based assays, and SYBR Green I dye chemistry. Note: the TAMRA dye is available only with the StepOnePlus System. TaqMan probe-based assays provide outstanding specificity and sensitivity, and SYBR Green I dye chemistry is an economical alternative for target ident
37、ification, initial screening assays, or assays that require only a few reactions. Software Analysis (Figures 7 and 8) The software for the StepOne Systems supports a variety of analysis methods, including: Absolute quantitation Standard curve Relative quantitation Relative standard curve Comparative
38、 CT (CT) Presence/absence (plus/minus) assays with an internal positive control Melt curve analysis Genotyping (including real-time amplification) 7 Figure 9. Melt Curve Analysis. 3.4 2.9 2.4 1.9 1.4 .9 .4 -.1 Derivative Reporter (-Rn) 65.070.075.0 Tm: 79.2 80.085.090.095.0 Melt Curve Temperature (C
39、) In this experiment, standard PCR was run to generate amplicons for 18S, -actin, and GAPDH. Aliquots of each amplicon were combined into one reaction tube and a melt curve analysis was performed. Multiplex TaqMan Assays on the StepOnePlus System showing amplification of cDNA (96 samples) using prob
40、es labeled with VIC and FAM reporters for 18S and TGF- target sequences respectively. 0.01 0.1 1 10 Rn 024681012141618202224262830323436384042 Multiplex Assays Cycle VeriFlex Blocks The 96-well StepOnePlus System features VeriFlex Block technology, which brings six independently control- lable pelti
41、er blocks together for precise temperature control and enhanced PCR functionality. VeriFlex Blocks deliver flexibility for those who have probes and primers that are optimized at different annealing temperatures. Results Because the StepOne and StepOnePlus Systems are factory- calibrated for optical
42、 and thermal accuracy, simply remarkable real-time PCR results are available right out of the box (Figures 9 and 10). Both systems can discriminate between two popula- tions of 5,000 and 10,000 template copies of a TaqMan Assay with 99.7% confidence. Both systems also demonstrate a linear dynamic ra
43、nge of 9 log units or more, as shown by the amplifica- tion plot (Figure 7). VeriFlex Block (96-well) Figure 10. Multiplex Assay. 8 StepOne System 48 wells for lower throughput 3 color for basic applications research Easily upgradeable to StepOnePlus System Systems At A Glance height: 51.2 cm (20.4
44、in) width: 24.6 cm (9.7 in) depth: 48.5 cm (19.1 in) depth: 48.5 cm (19.1 in) 2,024,000 4,000,000 300 Mouse 179,000 10,000 240 Rat 128,000 180 Drosophila melanogaster 38,000 50 Arabadopsis thaliana 95,000 40 Caenorhabditis elegans 90,000 60 Canine 6,000 N/A Rhesus macaque 70 N/A * Includes miRNA, ge
45、ne copy number, and mitochondrial assays Includes HapMap and drug metabolism genotyping assays Gene expression only Custom TaqMan Assays are available for any SNP, transcript, and genome TaqMan Gene Expression, SNP Genotyping, and MicroRNA Assays Applied Biosystems offers the most comprehensive set
46、of inventoried TaqMan Gene Expression and SNP Genotyping Assays available. More than 700,000 Gene Expression Assays and over 4.5 million pre-designed human, and 10,000 pre-designed mouse SNP Genotyping Assays are available at your finger- tips. Alternatively, you can submit your target DNA sequence
47、from any organism, and well custom-build an assay for you. Applied Biosystems also offers TaqMan MicroRNA Assays to quantitate miRNA with the sensitivity and specificity of TaqMan Assay chemistry. For more information on gene expression assays, visit ; for information on SNP Genotyping Assays, visit
48、 . TaqMan Assays Figure 11. The reagents ordering page in the Experimental Design Wizard. 11 Category/Product Description Quantity P/N Seals and Covers MicroAmp 48-Well Optical Adhesive Film 25 films 4375928 MicroAmp 48-Well Optical Adhesive Film 100 films 4375323 MicroAmp 96-Well Optical Adhesive F
49、ilm 25 films 4360954 MicroAmp 96-Well Optical Adhesive Film 100 films 4311971 Reaction Plates MicroAmp Fast Optical 48-Well Reaction Plate 20 plates 4375816 MicroAmp Fast Optical 96-Well Reaction Plate with Barcode (0.1 mL) 20 plates 4346906 MicroAmp Fast Optical 96-Well Reaction Plate with Barcode (0.1 mL) 200 plates 4366932 8-Well Strips MicroAmp Fast 8-Tube Strip, 0.1 mL 125 strips 4358293 MicroAmp Op