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上海圻明生物
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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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文献和实验光素只有异硫氰酸荧光素(FITC),丽丝胺罗丹明B200(RB200)。 2. 常用的荧光色素 (1)异硫氰酸荧光素 :又称异硫氰酸荧光黄,纯品为橙黄色粉末。分有结晶型与粉末型两种,结晶型荧光强度大、稳定、优于粉末型。分子式C21H11O2N5、分子量389,溶于水,易溶于pH8-9.5碳酸盐缓冲液中。最大吸收波长为495 nm。最大发射波长520 nm。异硫氰酸荧光黄性质稳定,于低温下可保存二年以上,但久置标记抗体能力弱,荧光亮度差,故应采用新产品。异硫氰酸荧光素借助
分别为0.3%双氧水和0.3%Triton X-100混合而成。配制方法是先用微波加热的36 ml PBS,再接着加120 ul Triton X-100,并加热一会儿,冷却至临用前加0.4 ml 30%H2O2。4、5%羊血清或封闭血清,用PBS稀释。5、含0.03%H2O2的0.05%DAB(避光):用20×DAB(1%,10 mg/ml)5 ul+0.1 ul 30% H2O2+95 ul PBS。6、一抗用PBS稀释(也可用抗体稀释液),二抗用中杉金桥的SP染色试剂盒。7、Xylene、梯度
免疫组化技术已经渗透到医学研究的各个领域,它在蛋白抗原的细胞准确定位上是其它蛋白检测方法无与伦比的。然而,要做好一张漂亮的染色切片,也不是十分容易的事。 我做免疫组化方面的实验多年,积累了很多经典案例。下面我把这些案例列举出来,并附上我们发现的问题以及如何解决这些问题的解决方法,以供大家实验参考和实验交流。 实验背景 以前做免疫组化,我一直用中杉金桥的 SP 三步法染色试剂盒,效果不错。在奥运会期间我准备做同批实验分不同批次酶免疫组化实验。第一批组化实验结果很好
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