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Lysozyme

溶菌酶(N-乙酰甘露糖聚糖水解酶)

Company: Roche Diagnostics
Catalog#: 10837059001
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Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE Combined with Primer Extension
Author:
Date:
2018-03-05
[Abstract]  In this assay, 3’ RACE (Rapid Amplification of cDNA 3’ Ends) followed by PE (primer extension), abbreviated as 3’ RACE-PE is used to identify the mRNA 3’ ends. The following protocol describes the amplification of the mRNA 3’ ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5’ end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3’ RACE. The conventional PE can only be used to locate the 5’ end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5’ → 3’ direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3’ end of the ... [摘要]  在该测定中,使用缩写为3'RACE-PE的3'RACE(cDNA3'末端快速扩增),随后是PE(引物延伸),以鉴定mRNA3'末端。以下方案描述E中半乳糖操纵子的mRNA 3'末端的扩增。并通过PE对相应的PCR产物进行可视化。在PE中,使用[γ-(32)P] ATP和T4多核苷酸激酶对确定的引物进行5'末端标记,其退火至3'RACE的PCR产物内的特定DNA分子。由于逆转录酶(RTase)仅在5'→3'方向聚合,常规PE只能用于定位mRNA转录物的5'末端。因此,使用Taq聚合酶代替RTase,进行PCR。因此,我们能够使用此测定法定位mRNA的3'末端。通过在变性8%尿素-PAGE(聚丙烯酰胺凝胶电泳)凝胶中分离DNA产物,可以直接显示和定量3'末端的相对量。 3'末端的确切位置可以通过比较这些最终的DNA产物与相应的DNA测序阶梯进行测序。


【背景】mRNA 3'末端的合成是E中的重要步骤。产生稳定的信使RNA(mRNA)的大肠杆菌。在真核细胞中,mRNA 3'末端形成是通过从内部磷酸二酯键切割,然后加入聚(A)尾;而在原核细胞中,通过终止转录或通过加工初级转录产生mRNA的3'末端(Altman和Robertson,1973; Nudler和Gottesman,2002; Zhao等人,1999年)。因此,分析mRNA ...

Isolation of Heterocysts from Anabaena sp. PCC 7120
Author:
Date:
2015-04-20
[Abstract]  During combined nitrogen step-down, filaments of cyanobacterium Anabaena sp. PCC 7120 differentiate about 5-10% of vegetative photosynthetic cells into heterocysts, the specialized cells for N2 fixation (Walk, 1996). Heterocysts have a thick cell wall reducing permeation of O2 and consist of two additional layers composed of glycolipids and polysaccharides. The difference in structure and composition of the cell wall between heterocysts and vegetative cells allows separation and isolation of heterocyst. Heterocysts isolated by this protocol can be subjected to protein analysis and activity measurements, which do not require strict anaerobic conditions. [摘要]  在组合氮降压期间,蓝细菌的鱼腥藻属 PCC 7120将约5-10%的营养光合细胞分化成异种细胞,这是用于N sub2固定的特化细胞(Walk,1996)。 杂合体具有厚的细胞壁,减少O 2的渗透,并且由两个由糖脂和多糖组成的附加层组成。 杂合细胞和营养细胞之间的细胞壁的结构和组成的差异允许异种细胞的分离和分离。 通过该方案分离的杂合体可以进行蛋白质分析和活性测量,其不需要严格的厌氧条件。

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