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- 详细信息
- 文献和实验
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- 保存条件:
低温
- 保质期:
详见说明
- 库存:
99
- 供应商:
上海圻明生物
- 规格:
2μg
精氨酸支原体PCR阳性对照质粒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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文献和实验网友eming43的经验 支原体的检测. PCR法 原理 该法是通过PCR技术将支原体16srRNA基因特异性扩增,来检测培养细胞中污染的支原体。PCR引物选自16sr RNA基因上的支原体特异片段,此片段与其他细菌的序列无交叉性杂交反应。扩增产物用琼脂糖凝胶电泳,经溴乙啶(EB)染色后在紫外透射仪上进行检测。 u 16sr DNA引物用水稀释至40umol/L。 (1).正向引物:5’-ACTCCTACGGGAGGCAGCAGTA-3’ (2).反向
类物质、部分种类需要精氨酸、O 2或葡萄糖,每一种支原体都有自身持点。多数支原体适合于偏碱条件下生存(PH7.6—8.0),对酸耐受性差。对热比较敏感,对一般抗生素不敏感。 支原体污染细胞后。培养液可不发生混浊。多数情况下细胞病理变化轻微或不显著,细微变化也可由于传代、换液而缓解。因此易被忽视。但个别严重者,可致细胞增殖缓慢。甚至从培养器皿脱落。 为确定有无支原体污染可做如下检酗: ①相差显微境检测:将细胞按种于事先放置于培养瓶内的盖玻片上,24h后取出,用相差油镜观察。支原体呈暗色微小
或者 Ct 值偏大的样本孔,到底是真的没有要检测的对象,还是扩增有问题,亦或是提取中的问题?为了发现这种假阴性的发生,合理的阳性对照可以监控实验的不同步骤: 扩增对照 Amplification Control 可使用含有扩增片段的质粒、假病毒或者基因组 DNA/cDNA 作为扩增阳性对照,监控荧光定量 PCR 的体系是否正常,包括酶、引物、探针等。如果检测中包含多个目标片段,可以将这些目标片段都克隆到同一个质粒中,方便制备和使用。当扩增对照没有扩增,或者 Ct 值大于预期,则说明定量 PCR
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