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- 详细信息
- 文献和实验
- 技术资料
- 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
中间普雷沃菌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
颗粒可含48000 拷贝,因而由 其造成的污染是一个值得特别重视的问题。 4、实验室中克隆质粒的污染:在分子生物学实验室及某些用克隆质粒做阳性对照的 检验室,这个问题也比较常见。因为克隆质粒在单位容积内含量相当高,另外在纯化 过程中需用较多的用具及试剂,而且在活细胞内的质粒,由于活细胞的生长繁殖的简 便性及具有很强的生命力。其污染可能性也很大。 二、污染的监测 一个好的实验室,要时刻注意污染的监测,考虑有无污染是什么原因造成的污染,以 便采取措施,防止和消除污染。 1、阳性对照:在建
一家小公司的产品,不过实验室一直用,效果一直有保证,同期进行连接的其他同学也都连接成功。 结果: 转化后,平板上均有数量不等的菌落产生,少时4-5个,多时10来个。摇菌扩增8 h,进行菌液PCR检测(含阳性对照),检测组无阳性结果,但一直在1000 bp左右出现较弱条带,阳性组正常。提取质粒后,10 μl体系双酶切2 h,竟然在2000 bp和3000 bp处出现较弱条带(比Marker稍暗)。 特此悬赏10个叮当,请高手指点迷津。
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