| Contents | BrdU Reagent,Fixing SolutionPrediluted Anti-BrdU DetectorPeroxidase Goat Anti-Mouse IgGConjugate DiluentSubstrate50X Plate Wash ConcwentrateStop Solution |
|---|---|
| Application | BrdU Cell Proliferation Assay |
BrdU Proliferation Assay involves incorporation of BrdU into cells cultured in microtiter plates using the cell layer as the solid phase. During the final 2 to 24 hours of culture BrdU is added to wells of the microtiter plate. BrdU will be incorporated into the DNA of dividing cells. To enable antibody binding to the incorporated BrdU cells must be fixed, permeabilized and the DNA denatured. This is all done in one step by treatment with Fixing Solution. Detector anti-BrdU monoclonal antibody is pipetted into the wells and allowed to incubate for one hour, during which time it binds to any incorporated BrdU. Unbound antibody is washed away and horseradish peroxidase-conjugated goat anti-mouse antibody is added, which binds to the Detector Antibody.
The horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of incorporated BrdU in the cells. The colored reaction product is quantified using a spectrophotometer.
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逆转录(reverse transcription)是以RNA为模板合成DNA的过程,即RNA指导下的DNA合成。此过程中,核酸合成与转录(DNA到RNA)过程与遗传信息的流动方向(RNA到DNA)相反,故称为逆转录。逆转录过程是RNA病毒的复制形式之一,需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现,是对中心法则的重要修正和补充。人们通过体外模拟该过程,以样本中提取的mRNA为模板,在逆转录酶的作用下,合成出互补的cDNA,构建cDNA文库,并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
1.模板提取(一般为RNA):Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定:分光光度计或NanoDrop
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBR Green Mix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBR Green,并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
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大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊,求推荐!

