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Ophiocordyceps Sinensis冬虫夏草源性成分探针法荧光定量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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文献和实验一种用于肝素粗品质量监测的、检测原料药中反刍动物DNA的多重RT-PCR分析方法的开发
动物源污染的实时荧光定量PCR技术。该检测法由一套针对反刍动物(牛、绵羊、山羊)和猪的冻干引物、探针及扩增内标组成。该方法经过两处分析机构验证:第一个位于FDA,第二个位于某州独立实验室。肝素钠多重实时荧光定量PCR(hMRTA)检测方法性能通过了美国FDA兽药中心研发处制定的特异性、灵敏度和专一性严格验收标准。符合早前建立的饲料中反刍动物原料多重实时荧光定量PCR检测法的灵敏度和重复性要求。hMRTA法检测猪源肝素钠粗品,98%达灵敏度,真阳性98%、假阴性2%。PCR检测法检测三个反刍动物物种,可作
基团(Quencher),其存在时可抑制信号基因的功能,不产生发光现象。 荧光PCR反应的实现 定量依据: 在PCR扩增特定基因时,反应体系中原始模板数愈多,到达平台期所需的循环数愈少;原始模板数愈少,到达平台期所需的循环次数愈多。 将不同浓度的原始模板数对PCR扩增到检测阈值所需的循环次数(Ct值)作图,便得到一个标准曲线 7700型定量PCR仪 Taq Man试剂盒(PE USA) 荧光定量PCR的过程 样品常规处理 → 加入反应管,设置阴,阳性对照 → PCR仪扩增 →电脑分析后给出
组DNA的引入,或通过引物设计避免非特异扩增。5. 溶解曲线不止一个主峰 引物设计不够优化:应避免引物二聚体和发夹结构的出现。 引物浓度不佳:适当降低引物的浓度,并注意上下游引物的浓度配比。 镁离子浓度过高:适当降低镁离子浓度,或选择更合适的 mix 试剂盒。 模板有基因组的污染:RNA提取过程中避免基因组DNA的引入,或通过引物设计避免非特异扩增。6. 扩增效率低 反应试剂中部分成分特别是荧光染料降解。 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
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