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低温
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- 供应商:
上海圻明生物
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50次
Luciferase 荧光素酶探针法荧光定量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superskyfly 我自己都觉得我自己很牛,千百人都做出来的试验,我就是做不出来。质粒用的是Promega p4.32-NF-kB-Luc, 转染用的是Invitrogen的Lipofactamin, 激活用的是Sigma生产的PMA和PHA-P,Assay用的是Promega dual,结果加了PMA和PHA的和不加的读数几乎没有区别。加了compound的和不加的也没有区别。 用过PC3, Jurkat, A549,HCT116都是一样。
荧光素酶作为一种报告基因要在检测时比较其相对发光强度值,就要在转染时引入另外一个报告基因进行转染条件、效率的标准化归一。Promega公司在这方面有双荧光素酶检测试剂盒,就是转染时同时转入两个质粒,包括萤火虫荧光素酶催化的反应和海肾(一种来自海洋的生物发光动物──三色堇)荧光素酶催化的反应。下面是说明书的一段原话:Firefly and Renilla luciferases,because of their distinct evolutionary origins
相关专题 Luciferase片段缺失实验 经检测发现多次裁切对荧光 素酶 表达量产生的影响相同(即检测组与空白组有明显差异,但各裁切的检测组间没有明显差异),故可推断转录调控位点应位于-282~近启动子端区域。 Luciferase定点突变 实验 对不同位点( 生物信息 学预测)分别进行点突变,LXRE2位点突变后的检测组与野生型的检测组间存在明显差异,而LXRE
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