![SMOBIO/[RQ2110] ExcelRT™ One-Step RT-qPCR Kit (TaqMan, ROX), 200 Rxn/TaqMan, ROX), 200 Rxn</span>
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Description
The ExcelRT™ One-Step RT-qPCR kit (TaqMan, ROX) is designed for reverse transcription and quantitative real-time analysis of a specific target RNA by one-step reaction. The ExcelRT™ One-Step RT-qPCR kit (TaqMan, ROX), consisting of One-Step RT Enzyme Mix and 2X One-Step qPCR Master Mix, is a convenient kit designed for highly efficient cDNA synthesis and highly specific real-time PCR in a single tube. The One-Step RT Enzyme Mix contains a thermostable ExcelRT™ Reverse Transcriptase and a RNAok™ RNase inhibitor. Consequently, One-Step RT Enzyme Mix can reverse transcribe RNA to cDNA at a wide temperature range from 42 to 60°C and active against RNase A, RNase B and RNase C. By containing specialized hot-start Taq DNA polymerase, which greatly reduce primer-dimer formation and can be activated within 2 minutes, the 2X One-Step qPCR Master Mix features high specificity and is suitable for fast cycle program. This master mix includes ROX reference dye for normalization of each RT-qPCR assay.
Features
High yield
Reverse transcription at wide temperature range (42-60°C)
High specificity
Suitable for fast cycle program
With ROX reference dye
Storage
Aliquot to avoid multiple freeze-thaw cycles (stable within 30 freeze-thaw cycles)
Protect from light
-20°C for 12 months
Reverse transcription at wide template range
ExcelRT™ One-Step RT-qPCR Kit can quantitative analyze target RNA from a wide range of RNA template input. The amplification plot of one-step RT-qPCR with total RNA templates ranging from 1 pg to 1 ng in quantity, analyzed by using RQ2110 ExcelRT™ One-Step RT-qPCR Kit (TaqMan, ROX) for RT-qPCR amplification.
Reverse transcription at wide temperature range (42-60°C)
ExcelRT™ One-Step RT-qPCR Kit can quantitative analyze target RNA at a wide temperature range (42-60°C). The amplification plot of one-step RT-qPCR with reverse transcription at temperature range from 42 to 60°C, analyzed by using RQ2110 ExcelRT™ One-Step RT-qPCR Kit (A) or kit from brand N (B) for RT-qPCR amplification. The overlapped amplification curves display that ExcelRT™ One-Step RT-qPCR Kit preforms successfully cDNA synthesis at wide temperature range.
Contents
Component | Volume |
One-Step RT Enzyme Mix | 400 μl |
2X One-Step qPCR Master Mix | 2 x1 ml |
Storage
Aliquot to avoid multiple freeze-thaw cycles (stable within 30 freeze-thaw cycles)
Protect from light
-20°for 12 months
Manual
Manual_RQ2110_ExcelRT™ 2X One-Step RT-qPCR Kit (TaqMan, ROX)
SDS
SDS_RQ2110
Recommended primer design
Amplicon size: 80-150 bp
Tm value: around 60°C (calculated with Primer3 software)
Primer length: 17-25 mer
Sequence:
‒ 45-55% of GC content isrecommended.
‒ Avoid regional high GC or ATcontent
‒ Avoid palindrome sequence
‒ Sequence with G or C at the 3" end is recommended.
Specificityof primers should be confirmed through a BLAST search.
Recommended probe design
Tm value: 6-10°C higher than primers
Probe length: 20-30 mer
Sequence:
‒ 35-65% of GC content is recommended.
‒ Avoid regional high GC or AT content
‒ Select the strand contains more C’s than G’s
‒ Avoid palindrome sequence
‒ Avoid a G at the 5" end to prevent quenching of the 5’fluorophore.
Specificityof probe should be confirmed through a BLAST search.
Recommended reaction mixture set up for qPCR
Component | Volume | Final concentration |
Template RNA | Varied | 1 pg – 1 μg |
Forward primer (10 μM) | Varied | 125 – 900 nM |
Reverse primer (10 μM) | Varied | 125 – 900 nM |
TaqMan Probe (10 μM) | Varied | 125 – 900 nM |
One-Step RT Enzyme Mix | 2 μl | 1X |
2X One-Step Master Mix | 10 μl | 1X |
ddH2O | to 20 μl | - |
Total volume | 20 µl | - |
*Template amount varies depending on the copy number of target present in the template solution.
** The PCR primer and probe concentration for an optimalqPCR reaction may vary according to primers’ and probe’s properties.
Recommended qPCRProgram
Standard program
Steps | Temp. | Time | Cycles |
Reverse transcription | 42°C - 60°C (45°C - 55°C is recommended) | 10min | 1 |
Enzyme activation | 95°C | 3 min | 1 |
Denaturation | 95°C** | 15 sec | 40-50 |
Annealing/ Extension | 60°C | 1 min |
Fast program
Steps | Temp. | Time | Cycles |
Reverse transcription | 42°C - 60°C (45°C - 55°C is recommended) | 5 min | 1 |
Enzyme activation | 95°C | 20 sec | 1 |
Denaturation | 95°C** | 3 sec | 40-50 |
Annealing/ Extension | 60°C | 30 sec |
[RP1000] ExcelRT™ Reverse Transcriptase
High yield
Thermostable, up to 50°C, during first strand synthesis
High processivity, generating cDNA up to 8 kb
Reduced RNase H ribonuclease activity
[RI1000] RNAok™ RNase Inhibitor
Application
RT-PCR
cDNA Synthesis
in vitro transcription
[RP1400] ExcelRT™ Reverse Transcription Kit II
Contains all components for reverse transcription
High yield
Thermostable, up to 50°C
Reduced RNase H ribonuclease activity
Suitable for real-time PCR
Contains all components for reverse transcription
High yield
Thermostable, up to 50°C
Reduced RNase H ribonuclease activity
Suitable for real-time PCR
[TQ2110] ExcelTaq™ 2X Q-PCR Master Mix (TaqMan, ROX)
High sensitivity
High specificity
With ROX reference dye
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向左转|向右转
如果是用转录出的cDNA做那跟平时就一样了。不需要特别的试剂盒、
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
和RevertAid™HMinusFirstStrandcDNASynthesisKit，FirstStrandcDNASynthesisKit
大家又没有用过，这个公司的酶据说很好啊。
逆转录（reverse transcription）是以RNA为模板合成DNA的过程，即RNA指导下的DNA合成。此过程中，核酸合成与转录（DNA到RNA）过程与遗传信息的流动方向（RNA到DNA）相反，故称为逆转录。逆转录过程是RNA病毒的复制形式之一，需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现，是对中心法则的重要修正和补充。人们通过体外模拟该过程，以样本中提取的mRNA为模板，在逆转录酶的作用下，合成出互补的cDNA，构建cDNA文库，并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
加尾法是采用加A酶先对mirna进行加尾，然后再用带oligodt的引物反转录，他的反转录引物是通用的，一次反转录可以获得所有miRNA的cdna，效率高。茎环法是采用特异性反转录引物序列+颈环结构作为反转录引物进行反转录的，一次反转录只能获得一种mirna的cdna，可能有几种一起反转录的，这个我不太清楚一起反转录的效果。然后茎环法的经典即ABI的探针法定量试剂盒。我用过茎环法反转录+染料法定量的，下游引物为通用引物，结果不太好，就现在用的这家的他采用的是双向引物都是特异性的，效果还可以。不知道我说的清楚不，希望能对你有帮助。
