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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
和评估状况相称的灵敏性。以下是一种使用实时聚合酶链式反应(PCR,hMRTA)测定粗品肝素中反刍动物污染物的分析方法。此法已使用猪源粗品肝素和牛标准物进行了适应性评估。可用于鉴别猪源粗品肝素中的反刍DNA。本草案提供了RT-PCR法检测猪粗品肝素中反刍原料的详细方法、所需试剂和设备。其他合适的替代方法也可进行资格验证,以用于检测粗品肝素中的反刍原料。 材料及方法 建议每批待测样品(提取、纯化和PCR)都设立阴性对照。并同时给每批Bio gX Ruminant
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