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- 供应商:
上海圻明生物
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2μg
人类嗜T淋巴细胞病毒通用RT-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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文献和实验基因编辑再次升级!领域大牛刘如谦 Cell 发文开发新工具,可安全高效进行体内基因编辑
,例如慢病毒和腺相关病毒等。然而,病毒作为载体会延长在转导细胞中的表达,这也就增加了脱靶的频率。 由于核糖核蛋白(RNPs)在细胞中的生命周期较短,因此直接传递 BE RNPs,而不是编码 BE 的 DNA 可以显著降低脱靶的频率。然而,在体内将 BE RNPs 传递到多个组织和器官的通用策略尚未被报道。 病毒样颗粒(VLPs)是一种可以感染细胞但不含病毒遗传物质的病毒蛋白质组合,已成为一种有潜力的载体,可以将基因编辑器 RNPs 传递给细胞。不过,到目前为止,现有的用于传递基因编辑器 RNPs
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
之前转染HEK293作为预实验,已经用WB证明有效,你之后的wb有没阳性对照呢或者说用HEK293作为对照呢 birkin chenhaombb wrote: 你用的什么载体啊?稳定转染的?怎么用G418筛选啊,费时费力,直接病毒转 用的pCMV6Entry,已经是慢病毒载体了……就是因为这种细胞转染率太低(据师姐说大概15%)不得已才做稳定转染,想说看看能不能让转染率上来一点
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