Product Highlights
- Sensitive – optimized buffer formulation delivers reliable quantification from even very low copy number RNA targets
- Reproducible – consistent results between technical replicates for increased confidence in results
- Robust – reliable detection of RNA targets from a broad range of sample types
- Fast – delivers reproducible, accurate assay results in as little as 40 minutes
- Efficient – excellent performance in multiplex assays
- Specific – antibody-mediated hot-start DNA polymerase minimizes non-specific amplification for improved assay sensitivity and reliability
Product Description
The SensiFAST Probe No-ROX One-Step Kit has been optimized for fast, efficient, unbiased cDNA synthesis and subsequent highly-sensitive, reproducible real-time PCR detection in a single tube. SensiFAST Probe One-Step has been optimized to deliver excellent results in both singleplex and multiplex assays.
An antibody-mediated hot-start DNA polymerase promotes rapid activation and supports highly-specific amplification, which in turn improves assay sensitivity and dynamic range. A combination of the latest advances in buffer chemistry and PCR enhancers confer superior assay performance under fast thermal cycling conditions. The inclusion of separate RiboSafe Inhibitor ensures accuracy by protecting RNA targets from RNase degradation.
The SensiFAST™ Probe No-ROX One-Step Kit has been validated on all commonly-used real-time instruments that do not require the passive reference dye ROX. SensiFAST Probe One-Step has been formulated for use with dual-labelled probes, including TaqMan®, Scorpions® and molecular beacon probes.
Applications
- Gene expression analysis
- Pathogen detection
- RNA viral pathogen detection
- Genetic profiling
- miRNA profiling / quantification
- Genetically modified organisms (GMO) characterization
Introduction to SensiFAST
Overview, features and benefits of the SensiFAST product familyReal-Time PCR Selection Chart
One-step Vs. Two-step real-time RT PCR
A discussion of the pros and cons of each detection strategy.ebiomall.com
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0.5 M tris-HCl (pH 6.8) 20%
丙三醇 20%
20%SDS 20%
0.1%溴酚兰 5%
2-巯基乙醇 10%
双蒸水 25%
不调PH值,PH在配0.5 M tris-HCl 时已调,缓冲液呈蓝紫色。
蛋白质上样缓冲液中的SDS和DTT分别可以屏蔽蛋白质电荷以及破坏二硫键,避免形成多聚体。
DNA上样缓冲液主要是起沉降和指示作用,不可用于蛋白,否则会影响蛋白相对分子量定量。
同样蛋白上样缓冲液中的tris-HCl浓度过高也会使溴酚蓝条带扭曲。
2乘SDS-PAGE上样缓冲液(20ml体系)
1.0mol/L Tris-HCL (pH 6.8) 2ml
1.0mol/L DTT 4ml
SDS 0.8g
溴芬蓝 0.04g
甘油 4ml
dd水 定容至20ml
4℃冰箱保存,可以不分装,我们实验室一般用三个月以上
先配1.0mol/L的DTT,双蒸无菌水溶解4℃冰箱保存,配上样缓冲液的时候加入就可以了,都混匀保存也没问题的
配制分三步: 1) 1 M Tris-HCl (pH 8.0) 50 ml的配制:称取Tris碱6.06 g,加超纯水40 ml溶解,滴加浓HCl约2.1 ml调pH至8.0,定容至50 ml。
电泳缓冲液的另一个作用是使溶液具有一定的导电性,以利于DNA分子的迁移,例如,一般电泳缓冲液中应含有0.01-0.04mol/L的Na+离子,Na+离子的浓度太低时电泳速度变慢;太高时就会造成过大的电流使胶发热甚至熔化。
电泳缓冲液还有一个组分是EDTA,加入浓度为1-2mmol/L,目的是螯合Mg2+ 等离子,防止电泳时激活 DNA酶,此外还可防止Mg2+离子与核酸生成沉淀。
蛋白电泳上样缓冲液包括2%SDS , 0.1%溴酚蓝 10%甘油,SDS是保证样品中的所有蛋白带电荷一致,减少电荷对电泳结果的影响。还原剂是让二硫键处于断开状态,保证蛋白分子的线性.
溴酚蓝作用是指示上样蛋白的跑胶时标记的,甘油是增加上样重量的,以免漂浮,煮沸是使蛋白变性的永久保存
最好是测浓度,一般不超过30-40ug,上样缓冲液一般4×或者5×,这个没太大区别。
1mol/l Tirs-HCl(PH8.8) 0.6ml
10%SDS 2ml
50%甘油 5ml
2-巯基乙醇 0.5ml
1%溴酚兰 1ml
水 0.9ml
另外:SDS不好溶解建议加热溶解,如果要做非还原SDS-PAGE请将2-巯基乙醇 0.5ml换成超纯水即可