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低温
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详见说明
- 库存:
99
- 供应商:
上海圻明生物
- 规格:
2μg
葡萄状穗霉属通用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。
3) 连接片段浓度比的问题 在阐明上述问题同时,本人尽可能举些实验中的问题案例予以说明。 一、克隆基因的酶切位点问题 1、克隆位点选择的问题。首先要对目标基因进行酶切位点扫描分析,列出其所含酶切位点清单。然后对照质粒多克隆位点,所选择的克隆位点必须是目标基因所不含的酶切位点。这是常识,不赘述。 2、保护碱基数目的问题。在设计PCR引物时,引入酶切位点后,常常要加入保护碱基,这是大家所熟知的。但是保护碱基数量多少,可能被新手所忽视。这种忽视碰
相关专题 我想表达遇到的第一个瓶颈估计就是为什么我的外源片段插到载体里面,PCR 鉴定没问题,双酶切也OK,可是就是不见表达。一般我们是如何判断没有表达呢?大多都是首先进行SDS-PAGE。在跑胶的时候一定要设对照,比较严谨的电泳对照,应该是:Marker,标准品阳性对照(如果有,且打算做Western的话),以及空白载体(诱导)和重组载体(不诱导)2个阴性对照,再加上诱导不同时间的表达结果。Marker用于判断条带大小,标准品用于判断
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