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- 供应商:
上海圻明生物
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50次
Fasciola gigantica大片吸虫染料法荧光定量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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如何获得高质量、高可信度的荧光定量PCR结果--高质量cDNA标准
跨内含子设计引物的方法来避免基因组DNA及假基因(Pseudogenes)的影响。如分别在相邻的两个外显子上设计qPCR引物,这样基因组DNA的扩增片段与cDNA扩增片段相比就增加了一个较大的内含子,而荧光定量PCR的延伸时间很短,很难扩增出大片段,如果使用SYBR Green方式检测,一旦有来源于基因组的扩增片段出现也可以在分析溶解曲线时发现。而对于原核生物,必须经过DNase I消化去除基因组DNA;但是,很多实验者发现,跨内含子设计引物有时的实验结果不佳,原因可能是基因组DNA增加了模板的复杂
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