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Manganese(II) chloride tetrahydrate

氯化锰(II)四水合物

Company: Sigma-Aldrich
Catalog#: M3634
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Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity
Author:
Date:
2017-06-20
[Abstract]  DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human Polθ is able to modify the 3’ terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3’ terminal ends of RNA and DNA for various applications in ... [摘要]  DNA聚合酶θ(Polθ)是一种混杂的酶,对易错的DNA双链断裂(DSB)修复途径而言是必需的,称为替代性末端连接(alt-EJ)。 在这种形式的DSB修复中,Polθ通过模板和非模板核苷酸添加循环在切割的DSB的3'末端处进行末端转移酶活性。 由于人Polθ能够用广泛的多种核糖核苷酸和脱氧核糖核苷酸类似物修饰DNA和RNA的3'末端,因此其末端转移酶活性对于生物技术应用比末端脱氧核苷酸转移酶(TdT)更有用。 在这里,我们详细介绍使用纯化的人Polθ修饰生物技术和生物医学研究中各种应用的RNA和DNA的3'末端的简单方法。
【背景】人类POLQ基因编码含有N末端超家族2(SF2)型解旋酶结构域和C末端A家族聚合酶结构域的大蛋白质(Sfeir和Symington,2015; Black et al。,2016; Wood and Doublie, 2016)。蛋白质也编码一个大的中心结构域,其功能尚未归入。 Polθ在后生动物中表达,已被证明在DNA复制和修复的多个方面起作用(Black et al。,2016; Wood and Doublie,2016)。最近的工作表明,哺乳动物Polθ对于易于识别的DNA双链断裂(DSB)修复途径而言是必需的,称为替代性末端连接(alt-EJ),也称微小鼠介导的终结合(MMEJ)(Yousefzadeh et al。 ,2014; ...

Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP
Author:
Date:
2016-08-20
[Abstract]  VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al., 2013). Assaying VPS34 activity ... [摘要]  VPS34是哺乳动物细胞中唯一的III类磷脂酰肌醇-3-激酶(PI3K),并且产生绝大多数细胞磷脂酰肌醇-3-磷酸[PI(3)P]。 PI(3)P是一个关键的信号脂质,在诸如内吞作用和自噬的过程中起许多膜运输的作用。 VPS34是关键的细胞调节剂,功能丧失可具有灾难性作用并且是胚胎致死的(Zhou等人,2011)。 细胞PI(3)P的水平可以通过荧光染色技术确定,并且可以用于监测对VPS34活性的影响,然而重要的是验证任何变化由VPS34介导,特别是作为PI(3)的替代途径, P生产是可能的,例如通过II类PI3K(Devereaux等人,2013)。 直接在体外测定VPS34活性可以是描绘特定刺激的作用的关键阶段。

Stable Transformation of Cyanobacterium Synechocystis sp.
Author:
Date:
2014-11-05
[Abstract]  Cyanobacteria are prokaryotes, which perform oxygenic photosynthesis. Among them, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a well characterized model system for studies on oxygenic photosynthesis, light signal transduction etc. Moreover, Synechocystis is applied in biotechnological applications (Desai and Atsumi, 2013). Stable transformation of Synechocytis is achieved via the uptake of DNA and incorporation into the host genome by homologous double recombination. This allows for the generation of gene knock-outs (KO) by replacing the coding sequence of the gene of interest by a KO-cassette (comprising of a selection marker flaked by sequences of the gene of interest) or stable overexpression of ... [摘要]  蓝细菌是原核生物,其执行含氧光合作用。其中,单细胞蓝细菌集胞藻 PCC 6803(下文称为"集胞藻")是用于研究含氧光合作用,光信号转导等的良好表征的模型系统。此外,集胞藻应用于生物技术应用(Desai和Atsumi,2013)。通过DNA的吸收和通过同源双重重组并入宿主基因组中实现集胞藻的稳定转化。这允许通过用KO盒(包含由感兴趣的基因的序列片段化的选择标记)或感兴趣的基因的某些基因的稳定过表达来产生基因敲除(KO)在相应的过表达盒插入宿主基因组上的中性插入位点之后。稳定转化集胞藻由Grigorieva和Shestakov(1982)报道。从那时起,初始协议的变体已经成功应用于变换集胞藻。在这里,我们描述了成功应用于集胞藻的稳定转化的实验室方案(Schwarzkopf等人,2014)。

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