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Turlock病毒染料法荧光定量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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文献和实验新型冠状病毒背景介绍与核酸检测整体解决方案分享,从核酸模板制备到 RT 实验策略,再到 qPCR 实验解析与应用。
了我的扩增曲线呢? 其实,这种现象并不是偶发的,有很多原因可能导致,并且有个专属名称「Hook Effect」,翻译过来叫「鱼钩效应」。「鱼钩效应」指在荧光定量 PCR 过程中,DNA 扩增到指数增长期后出现的一种荧光信号不能保持稳定(或者上升)而出现下降的现象 [1]。 导致扩增曲线出现这一现象的因素比较复杂,这里我们分别从嵌入性染料法与探针法两种定量方式的角度为大家解释: 嵌入性染料法: 嵌入性染料法(例如 SYBR Green I)产生「鱼钩效应」主要是由于模板序列混杂,在 PCR
实时荧光定量 PCR 技术(Quantitative Real-time PCR,简称 qPCR)是在 PCR 扩增过程中,通过实时监测荧光信号的变化,达到对待检测样本中初始模板定量分析的方法。当前 qPCR 技术被广泛应用于临床疾病诊断,动物疾病监测,食品安全分析等领域。随着 2020 年新冠疫情在全球的蔓延,qPCR 技术更是因其检测通量高,速度快,操作简便等优势成为新冠病毒核酸检测的有效方法被众人所熟知。 那么为了得到可靠、准确的检测结果,我们需要注意哪些方面呢?其实决定一次实验成功
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