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- 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
无齿类圆线虫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
检测时都可能检测到一些非目标条带。可以通过电泳检测酶切之前的质粒判断质粒的质量,排除可能的原因。 调整酶切温度、时间、buffer 体系等,可以解决酶切杂带或切不开的问题。 6. PCR 扩增不出条带/条带弱/非特异条带怎么办? ①检查模板 DNA 是否存在降解,此外模板的上样量过高或者过低,也会影响片段的扩增; ②重新设计引物,比如引物与非目标序列同源性高可导致非特异扩增或无扩增产物,引物自身形成发夹结构、引物二聚体从而影响模板结合等。可以借助专业性软件检查引物结构并重
liuruya 问别人要了个带GFP-tag(GFP-N2载体)的质粒,目的基因1.4kb,分子量约52kD,转染293T细胞能见荧光,WB杂不出来,设置了阴性和阳性对照证实不是WB的技术问题。后来又换了个flag-tag,仍然杂不出来WB。仔细检查过序列,不存在移码的问题,非常费解。有同学分析是空间位阻问题,不知换个tag是不是能解决这个问题。有同学遇到过这种问题么,或者帮忙分析一下可能的原因。谢谢!!! zhuqueleee
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