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你可能喜欢关于Taqman探针法中NTC也出现荧光信号，怎么回事呢(荧光信号,探针法,探针,阈值) - PCR实验 - 生物秀
标题: 关于Taqman探针法中NTC也出现荧光信号，怎么回事呢(荧光信号,探针法,探针,阈值)
摘要: [关于Taqman探针法中NTC也出现荧光信号，怎么回事呢(荧光信号,探针法,探针,阈值)] 我做的是Taqman相对定量，之前做目的基因的标准曲线的时候，NTC再30多个循环有起飞，但是当时标准品浓度较高，Ct值最大的也只有20多，所以没有在意NTC的问题，但是当做我的样品时，发现样品的Ct值都再３０多，和水的特别接近，那这样就有干扰，不能反映真实的情况，我先考虑可能是探针浓度太大（20nm），后来降低了一倍（100nm），水做模板的就在阈值之下了，当我重复之前的实验时，NTC又出现荧光 关键词:[荧光信号 探针法 阈值 探针 标准曲线 标准品 目的基因]……
我做的是Taqman相对定量，之前做目的基因的标准曲线的时候，NTC再30多个循环有起飞，但是当时标准品浓度较高，Ct值最大的也只有20多，所以没有在意NTC的问题，但是当做我的样品时，发现样品的Ct值都再３０多，和水的特别接近，那这样就有干扰，不能反映真实的情况，我先考虑可能是探针浓度太大（20nm），后来降低了一倍（100nm），水做模板的就在阈值之下了，当我重复之前的实验时，NTC又出现荧光信号了，又继续降低探针量（50nm）还是有信号。后来咨询过仪器公司的技术支持，说taqman探针再循环数过高会自发水解，建议我加大模板量，我是做的逆转录，cDNA浓度不可能提高很大呀，而且10倍的浓度差异才3点几个循环的差异，还是和水不能完全分开吧？我做看家基因就比较好，NTC都再阈值之下，这和探针合成的质量是否有关系呢？另外在里搜了下，有些说这种情况可能是污染，我的水和枪头还有都是新的了。特迷惑，下步不知道怎么做呢，请大家多多指教！
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电话：021-【图文】实时荧光定量PCR――TaqMan探针法及设计原则_百度文库
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实时荧光定量PCR――TaqMan探针法及设计原则
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你可能喜欢用Taqman探针法做qPCR，结果很差，求高手指导 - 实验交流 - 生物秀
标题: 用Taqman探针法做qPCR，结果很差，求高手指导
摘要: [用Taqman探针法做qPCR，结果很差，求高手指导] 用Taqman探针法做qPCR，引物和探针都是参考的文献来得，之前用这个引物做过简单的PCR，是单一条带，但做qPCR结果很差，见附件中的图。探针换另外一个公司合成以后，结果还是没有好转，体系变化了结果也没有变化，跪求高手指导呀Amplification Plot jpg 关键词:[探针 模板浓度 引物 条带]……
用Taqman探针法做qPCR，引物和探针都是参考的文献来得，之前用这个引物做过简单的PCR，是单一条带，但做qPCR结果很差，见附件中的图。探针换另外一个公司合成以后，结果还是没有好转，体系变化了结果也没有变化，跪求高手指导呀
Amplification Plot.jpg回复如果体系没有问题的话，就是探针不行呗，岂能相信文献回复像是没扩增出来，但曲线还连在一起，是不是你的模板浓度太高了啊回复
如果体系没有问题的话，就是探针不行呗，岂能相信文献 你哭着对他说，文献里都是骗人的，你不可能做出那结果。。哈哈回复
像是没扩增出来，但曲线还连在一起，是不是你的模板浓度太高了啊 模板浓度不高呀，20ng左右
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..........
生物秀是目前国内最具影响力的生物医药门户网站之一，致力于IT技术和BT的跨界融合以及生物医药领域前沿技术和成功商业模式的传播。为生物医药领域研究人员和企业提供最具价值的行业资讯、专业技术、学术交流平台、会议会展、电子商务和求职招聘等一站式服务。
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电话：021->>>>>CFX96 Touch& Real-Time PCR Detection System
Excitation and detection wavelength ranges for the CFX96 Touch& and CFX96 Touch Deep Well& systems.
Innovative Optical DesignThe CFX96 Touch& and CFX96 Touch Deep Well& systems' solid-state optical technology (six filtered LEDs and six filtered photodiodes) provides sensitive detection for precise quantification and target discrimination.
Scanning just above the sample plate, the optics shuttle individually illuminates and detects fluorescence from each well with high sensitivity and no cross talk. The optical system automatically collects data from all wells during data acquisition, so you can enter or edit well information on your own schedule.
Five-Target MultiplexingThe CFX96 Touch and CFX96 Touch Deep Well systems can discriminate up to five targets in a single reaction well. The optical filter sets are designed to maximize fluorescence detection for specific dyes in specific channels. At every position and with every scan, the optics shuttle is reproducibly centered above each well, so the light path is always fixed and optimal, and there is no need to sacrifice data collection in one of the channels to normalize to a passive reference.
Fast Scan OptionYou can complete SYBR& Green I and single-color FAM protocols even faster using the fast scan mode, which reads single-channel fluorescence from all 96 wells in just 3 seconds.
Fluorescence Resonance Energy Transfer (FRET)The CFX96 Touch and CFX96 Touch Deep Well systems' optics shuttle includes one channel with an LED-filter photodiode combination dedicated for FRET singleplex experiments, further expanding your experimental options.
If you're like most researchers, you want to get results faster. Many factors contribute to the overall time it takes to get PCR results: the time to design, program, and optimize a protocol, the time to run the protocol, and the time to analyze the results. Bio-Rad engineers have developed a complete thermal cycling system that will speed every step of the PCR process to give you what's really important & a shorter time from setup to results.
Protocol autowriter & generates an optimal protocol based on your polymerase, primers, and product length.
Thermal gradient feature & identifies optimal annealing temperature in a single run.
Reduced-mass sample block & fast ramping and settling produce the shortest time to target temperature available in a thermal cycler.
Intuitive software & makes programming faster and easier for PCR and real-time PCR.
The patented* reduced-mass sample block heats and cools more quickly than standard blocks, so average ramp rates are increased and overall run times are reduced.
Superior Uniformity Precision of the temperature steps is critical for the rate and efficiency of PCR. To obtain reliable, consistent results, all sample wells must maintain proper temperature throughout each incubation step. The CFX96 Touch& system uses six independently controlled thermal electric modules (TEs), the heating and cooling elements of the thermal cycler, to maintain tight temperature uniformity at all points during a run & even while ramping.
Rapid Arrival at Target Temperature A key component of overall protocol run time is the time required to reach target temperature, which is determined by the average ramp rate and the time needed for the sample block to reach thermal uniformity. Maximum ramp rate is less important because it can fluctuate significantly during the ramp. The CFX96 Touch system produces high average ramp rates and tight uniformity during ramping to yield fast time to target temperature and faster protocol run times. Run times can be dramatically shortened & to less than 30 min & while still producing accurate quantitative results. Now you can tailor your runs around your schedule instead of tailoring your schedule around your runs.
Superior uniformity with rapid arrival at target temperature. 1000-series thermal cyclers exhibit high average ramp rates, rapid settling time, and tight thermal uniformity throughout the ramp. This graph shows the temperature measured by probes in 15 wells across a sample block. The traces are nearly indistinguishable due to the tight uniformity. Note the consistent high average ramp rate throughout heating and cooling.
* U.S. patent 7,632,464.
Efficient Optimization Determining the optimal temperature for primer annealing is crucial for efficient and specific amplification of product. With the CFX96 Touch systems' thermal gradient feature, you can determine the optimal temperature for primer annealing in a single experiment, minimizing the use of precious samples and reagents and saving valuable research time. At any step in a protocol, you can program a temperature gradient of up to 24&C across the reaction block. The thermal cycler provides exceptional temperature uniformity and reproducibility within each gradient zone, and the temperatures can easily be programmed and viewed on the touchscreen or onscreen in the software so you can quickly identify the optimal incubation temperature.
Thermal gradient experiment for optimizing annealing temperature. A tenfold dilution series (106 to 10 copies) of plasmid containing GAPDH template was amplified in the presence of SYBR& Green dye using a protocol with an annealing thermal gradient ranging from 55 to 68&C. Results are presented for three temperatures, showing 62&C as the optimal in this case, with early quantification cycle (Cq) values and the highest standard curve efficiency. RFU, relative fluorescence units.
O-ring hermetic seal extends the life of the heating and cooling elements. Condensation from constant heating and cooling can degrade thermal electric modules (TEs), causing them to fail.
Bio-Rad 1000-series cyclers include a patented* O-ring seal (red in illustration) that forms an airtight barrier around the TEs, preventing exposure to condensation.
This substantially lengthens the life of TEs and guarantees outstanding thermal performance.
* U.S. patent 7,051,536.
The Ultimate in Configuration Flexibility
A System That Grows with Your ResearchThe flexibility of the 1000-series platform allows your setup to grow and evolve as your needs change. Adding more instruments to increase throughput or expand options is as easy as connecting a USB cable. CFX Manager& software can independently run four instruments, including a combination of CFX96 Touch&, CFX96 Touch Deep Well&, or CFX384 Touch& real-time instruments and C1000 Touch& or S1000& thermal cyclers. You may start a run on one instrument while another is running a different experiment, or you can start the same experiment on all four instruments at once.
Configuration Options
Stand-Alone
Stand-alone thermal cyclers and real-time PCR detection systems
Stand-Alone Multi-Instrument
C1000 Touch cycler control of up to 3 S1000 cyclers
CFX96 Touch, CFX96 Touch Deep Well, or CFX384 Touch system control of up to 3 S1000 cyclers
Software-Controlled Stand-Alone
C1000 Touch cycler or CFX96 Touch, CFX96 Touch Deep Well, or CFX384 Touch system controlled by CFX Manager software
Software-Controlled Multi-Instrument
Up to 8 C1000 Touch cyclers, each connected to 1&3 S1000 cyclers, controlled by CFX Manager software
1 CFX96 Touch, CFX96 Touch Deep Well, or CFX384 Touch system connected to 1&3 S1000 cyclers, controlled by CFX Manager software
Up to 4 CFX96 Touch, CFX96 Touch Deep Well, and/or CFX384 Touch systems controlled by CFX Manager software
Excellent uniformity. IL-1& plasmid template diluted to 105 copies/reaction amplified in the presence of a FAM-labeled detection probe with iQ& supermix. Graph shows 96 replicates of 10 &l reactions. Average quantification cycle (Cq) = 19.81 & 0.10. RFU, relative fluorescence units.
Confidently analyze data from a broad range of sample concentrations even when multiplexing five targets. A&E, fluorescence data from a series of tenfold dilutions of plasmid DNA (108&102 copies) amplified using reporter dyes to monitor five targets: , FAM/ , HEX/GAPDH; , Texas Red/ , Cy5/ , Quasar 705/IL-1&; F, standard curves generated from data in A&E, reaction efficiencies range from 97 to 103%.
RFU, relative fluorescence units.
qbasePLUS Software
and Bio-Rad have teamed up to offer a powerful suite of tools that will speed up real-time PCR data analysis and accelerate discovery.
Key features of qbasePLUS software:
Reliable validation & based on proven solutions for quality control, normalization, and inter-run calibration
Efficient data analysis & import and consolidate information from multiple runs and multiple instruments to quickly analyze your complete data set, and use a guided statistical wizard to determine significance
Streamlined publication submission & export an RDML file containing annotations, such as sample and assay information, to conform to the MIQE guidelines
Instrument Compatibility of PCR Plastic Consumables
MiniOpticon& System
iCycler iQ&, iQ&5,MyiQ&, and MyiQ&2 Systems
Chromo4& System
DNA Engine Opticon& 2 System
CFX96 Touch&, CFX96 Touch Deep Well&, CFX384Touch&, and CFX Connect& Systems
High-profile individual tubes
CFX96 TouchDeep Well only
Low-profile tube strips
Hard-Shell& low-profile 96-well semi-skirted PCR plates
Hard-Shell high-profile 96-well semi-skirted PCR plates
CFX96 TouchDeep Well only
Hard-Shell low-profile 96-well skirted PCR plates
Multiplate& high-profile unskirted PCR plates
CFX96 TouchDeep Well only
Multiplate low-profile unskirted PCR plates
iQ& 96-well semi-skirted PCR plates
Hard-Shell 384-well skirted PCR plates
Optical flat cap strips
Microseal& 'B' adhesive seals
Microseal& ?C? optical seals
PX1& PCR plate sealer
Optically clear heat seal
is a comprehensive source of information on real-time PCR. Complete with detailed information on optimizing, validating, and analyzing real-time PCR data, this technically focused applications guide is a resource for experienced researchers as well as those wanting to learn more about using real-time PCR for applications such as qPCR, RT-qPCR, genotyping, and GMO detection. This guidebook includes chapters that cover:
Key concepts of real-time PCR
Getting started & experimental design
Multiplexing considerations
Data analysis
Gene expression analysis
Genotyping allelic discrimination
GMO detection
This guidebook comes with an easy-to-review real-time PCR quick guide to use at the bench. The quick guide covers the fundamentals of qPCR, design, optimization, and data analysis of qPCR experiments.
The CFX96& Optical Reaction Module converts the C1000 Touch& Thermal Cycler chassis into the powerful and precise CFX96 Touch Real-Time PCR Detection System. This six-channel system combines advanced optical technology with precise thermal control to deliver sensitive, reliable detection. Quickly set up runs and monitor amplification traces in real time on the integrated LCD touch screen. With up to five-target detection, unsurpassed thermal cycler performance, unrivaled stand-alone functionality, and powerful yet easy-to-use software, the CFX96 Touch System is designed to advance your qPCR.
With the CFX96 Touch Real-Time PCR Detection System you can:
Set up your system quickly & easy installation and factory-calibrated optics
Minimize sample and reagent use & up to 5-target multiplexing with sample volumes as low as 10 &l
Optimize reactions in a single run & thermal gradient feature
Analyze data faster & visualize all run data at once and export only the data you need in the format you want
Use advanced data analysis tools & normalized gene expression using
Configure the system to fit your needs & run without a computer, run up to 4 systems from 1 computer, or integrate with the CFX Automation System II for higher throughput
Combine the CFX96 Touch System with good laboratory practice standards & use
for data collection and analysis to simplify compliance with U.S. FDA 21 CFR Part 11 regulations
works with all CFX Real-Time PCR Detection Systems to enable walk-away, high-throughput qPCR operation. Integrate this robotic plate handler with one or two CFX Systems to maximize throughput while maintaining a compact footprint.
The expert design and wet-lab validation of all PrimePCR Assays ensure optimal assay performance. The integration of CFX Manager Software with PrimePCR Assays aids in the analysis and interpretation of experimental results.
Begin analyzing data in a single step by importing target, reference, and control information from a PrimePCR run file directly into CFX Manager Software
Combine data from multiple plates into a Gene Study to rapidly screen large numbers of targets or samples
Use powerful data visualization tools, such as hierarchical clustering and color-matched expression levels, to identify individual targets or clusters to consider for further investigation
offers various service plans to keep your systems functioning optimally
certify system installation and validate system performance
test temperature accuracy and uniformity
quickly and easily verifies the performance of your CFX Real-Time PCR Detection Systems
Thermal Cycler
C1000 Touch&
Maximum ramp rate, &C/sec
Average ramp rate, &C/sec
Heating and cooling method
Heats up to 105
Temperature
Accuracy, &C
&0.2 of programmed target at 90&C
Uniformity, &C
&0.4 well-to-well within 10 sec of arrival at 90&C
Operational range, &C
Programmable span, &C
Optical Detection
Excitation
6 filtered LEDs
6 filtered photodiodes
Range of excitation/emission wavelengths, nm
Sensitivity
Detects 1 copy of target sequence in human genomic DNA
Dynamic range
10 orders of magnitude
All channels, sec
FAM/SYBR& Green only, sec
Operating systems
Windows 7, Windows 8
Multiplex analysis
Up to 5 targets per well
Licensed for real-time PCR
Sample capacity, wells
Sample size, &l
1&50 (10&25 recommended)
Communication interface
Electrical approvals
Dimensions (W x D x H), cm/in
33 x 46 x 36 /13 x 18 x 14
Weight, kg/lb
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Modular thermal cycler platform, includes C1000 Touch& Thermal Cycler chassis, CFX96& Optical Reaction Module, CFX Manager& Software, qbase+ Software license, cables, reagents, consumables
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96–deep well optical module for real-time PCR, includes CFX Manager& Software, qbase+ Software license, cables, reagents, consumables, for use with C1000 Touch& Thermal Cycler Chassis
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96-well optical module for real-time PCR, includes CFX Manager& Software, qbase+ Software license, cables, for use with C1000 Touch& Thermal Cycler chassis
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Description
Instruction Manual, CFX96 Touch, CFX96 Touch Deep Well, CFX Connect, and CFX384 Touch Real-Time PCR Detection Systems, Rev E
CFX96 Touch and CFX96 Touch Deep Well Real-Time PCR Detection Systems installation Quick Guide, Rev C
CFX Qualification Plate & 96-Well Plate Format Product Insert, Rev B
CFX Manager& Software Protocol for the CFX96 System, Quick Guide, Rev C
CFX Manager Software Plate for the CFX96 System, Quick Guide, Rev C
CFX Manager Software Data Analysis for the CFX96 System, Quick Guide, Rev C
CFX Manager Software Gene Expression Analysis for the CFX96 System, Quick Guide, Rev D
Checking Firmware and Software Updates for the C1000 Touch Thermal Cycler and the CFX96 Touch, CFX96 Touch Deep Well, and CFX384 Touch Real-Time PCR Detection Systems, Quick Guide, Rev C
Plate Preparation for the CFX384 Real-Time PCR Detection System Using a Tecan Robotics System, Rev A
Plate Preparation for the CFX384 Real-Time PCR Detection System Using the IDEX Health & Science Innovadyne Dispenser, Rev A
CFX96 Touch& Real-Time PCR Detection System Flier, Rev A
CFX96 Touch Real-Time PCR Detection System Brochure, Rev D
CFX96 Touch Real-Time PCR Detection System Specifications Sheet, Rev C
CFX Manager Software, Security Edition Product Information Sheet, Rev A
1000-Series Thermal Cyclers Brochure, Rev C
Amplification Plastics and Reagents Selection Guide, Rev B
Gene Expression Analysis Solutions Brochure, Rev C
High-Throughput Real-Time PCR Solutions Brochure, Ver B
Real-Time PCR Applications Guide, Rev B
A Practical Approach to RT-qPCR &&Publishing Data That Conform to the MIQE Guidelines, Ver E
Optical Design of CFX96 Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye, Rev A
Real-Time qPCR as a Tool for Evaluating RNAi-Mediated Gene Silencing, Rev A
Fast Optimization of a Multiplex Influenza Identification Panel Using a Thermal Gradient, Rev A
A Practical Guide to High Resolution Melt Analysis Genotyping, Rev A
High Resolution Melt Parameter Considerations for Optimal Data Resolution, Rev A
A Fast Semi-Automated Quantitative PCR Method for Monitoring Differential Gene Expression for Drug Target Discovery, Ver B
Gene Expression Real-Time PCR Workflow Flier, Ver A
Protocol for Single Cell Gene Expression Profiling by Multiplex RT-qPCR, Ver A
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