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SARS-CoV-2冠状病毒(新冠病毒)S基因探针法荧光定量RT-PCR试剂盒上海圻明生物优势供应。更多产品资料欢迎免费咨询。
One of the many important uses of PCR technology is that it can be used to label DNA probes with high specific activity. PCR technology has high specificity, and can synthesize probe DNA fragments in quantities within 1~2h if [α-32P]dNTP or other markers are added to the substrate
dNTPs, the probe DNA can be well labeled during DNA synthesis, and the incorporation rate of the marker can be as high as 70%~80%. Therefore, PCR labeling technology is particularly suitable for large-scale detection and non-radiolabeling. The disadvantage of this method is that a specific pair of PCR primers is synthesized.
Labeling can also be achieved by using small fragments prepared from probe DNA as primers.
Solution preparation
1. Prepare a stock solution
Unless otherwise stated, all unused stock solutions should be divided into disposable aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1.1* Acid Stock Solution (125X):
Add 20 μL DMSO to *ate (component B) to make a 125X* acid stock solution.
2. Prepare standard solutions
*Salt standard solution
Add 50 μL of 1 mM KH2PO4 (Component C) to 950 μL of deionized water or enzyme reaction buffer to give a 50 μM * saline standard solution (PS7). A 50 μM * saline standard solution (PS7) was taken and serially diluted 1:2 to obtain a serially diluted phosphate standard with deionized water or enzyme reaction buffer.
3. Prepare a working solution
Add 20 μL of 125X* stock solution to 2.5 mL of sterile H2O and mix well to make a working solution of *salt. Avoid potential Pi contamination. Note: Avoid direct exposure of *salts (component B) to light. Due to the high sensitivity of this assay to Pi, it is extremely important to use Pi-free labware and reagents.
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文献和实验许石正丽的这篇综述可以解释:为什么每次冠状病毒疫情都会打得我们措手不及?
(SARS-Cov-2),短时间内在国内造成严重疫情。回顾历次冠状病毒疫情,广泛的流行病学调查以及对冠状病毒起源和演变的认知,使我们对冠状病毒有了更深的了解。不断地变异和重组使其爆发似乎在意料之中,然而每次疫情的严重性却常在科研人员以及政府管理部门意料之外!究其原因,是否我们对冠状病毒的重视程度还不够?图片来源:美国国家过敏与传染病研究所2019 年 3 月,中科院武汉病毒研究所石正丽研究员在 Nature Reviews 发表综述文章《Origin and evolution
Real-time qPCR 手册——手把手教你从菜鸟到高手
反应条件不够优化:可适当降低退火温度或改为三步扩增法。 反应体系中有 PCR 反应抑制物:一般是加入模板时所引入,应先把模板适度稀释,再加入反应体系中,减少抑制物的影响。 7. 同一试剂在不同仪器上产生不同的曲线,如何判断? 判断标准:扩增效率,灵敏度,特异性 如果扩增效率在 90%-110%,都是特异性扩增,都可以把数据用于分析。 8. 扩增曲线的异常?比如「S」型曲线? 参比染料设定不正确 (MasterMix 不加参比染料时,选 NONE) 模板的浓度
水至25 ul。 混匀,置于SLAN荧光定量PCR仪中。95℃5min预变性后,95℃15 s→65℃35 s(荧光检测),40 cycles。荧光定量PCR一般把退火和扩增设成一个温度,只在扩增出现问题时才会考虑设梯度。 (侯哥论文中的:95 °C 10分钟→95 °C 15秒→60 °C 1分钟;40个循环。) 以双△Ct值法计算靶基因相对表达水平: GRP78相对表达水平=2-△(△CT
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