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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
鼻气管炎鸟杆菌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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文献和实验臂区域如果 GC 含量过高过低,或者有重复序列,都可能在核酸外切酶切割后形成发卡结构,影响重组效率,可以使用质粒设计软件来检查同源重组臂的设计。 ⑤插入序列本身的问题:有些序列本身存在连接载体困难、连接产物不稳定、连接产物在大肠杆菌中复制困难等问题。对于这类序列可以考虑将序列打断后分步构建,或者更换载体或感受态的方式尝试解决。 2. 质粒提取量不足 质粒提取量少常见的原因可能是: ①摇菌时间过长:大肠杆菌生长已经超过对数期,细菌老化,导致细胞和 DNA 降解。 ②摇菌时间不足:细菌生长不充分
或凹陷脱磷需CIP0.5u/pmolDNA末端。2、连接质粒载体和DNA插入片断时:(1)初次实验的可以建立几种不同的连接体系,比如插入片段与载体分子数比率可以为3:1,1:1,或1:3。(2)可以尝试三种温度和时间的连接如4度,过夜;15度,4-6小时;25度,1小时。3、细菌转化和筛选时:(1)阳性对照:如果是感受态细胞,-80度储存的在5-6周后,转化率低。可用一已知标准闭环质粒鉴定感受态细胞的转化能力。(2)阴性对照:用无DNA转化物的感受态细菌铺板,如果有菌落生长,说明其中抗生素浓度不够
立PCR 反应实验室及一般的检验单位都应设有PCR 阳性对照,它是PCR 反应是否成功、产物条带位置及大小是否合乎理论要求的一个重要的参考标志。阳性对照要选择扩增度中等、重复性好,经各种鉴定是该产物的标本,如以重组质粒为阳 性对照,其含量宜低不宜高(100 个拷贝以下)。但阳性对照尤其是重组质粒及高浓度阳 性标本,其对检测或扩增样品污染的可能性很大。因而当某一PCR 试剂经自己使用稳 定,检验人员心中有数时,在以后的实验中可免设阳性对照。 2、阴性对照:每次PCR 实验务必做阴性对照。它包括①标本
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