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Ethylenediaminetetraacetic acid disodium salt dihydrate

乙二胺四乙酸二钠盐二水合物

Company: Sigma-Aldrich
Catalog#: E5134
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Two Different Methods of Quantification of Oxidized Nicotinamide Adenine Dinucleotide (NAD+) and Reduced Nicotinamide Adenine Dinucleotide (NADH) Intracellular Levels: Enzymatic Coupled Cycling Assay and Ultra-performance Liquid Chromatography (UPLC)-Mass Spectrometry
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Date:
2018-07-20
[Abstract]  Current studies on the age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD+) levels decrease in an expected physiological process. Recent studies have shown that a reduction in NAD+ is a key factor for the development of age-associated metabolic decline. Increased NAD+ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging.

Given the importance of monitoring cellular NAD+ and ...
[摘要]  目前关于代谢功能障碍和虚弱的年龄相关发展的研究每天都有更多的证据。众所周知,随着衰老的进展,烟酰胺腺嘌呤二核苷酸(NAD + )水平在预期的生理过程中降低。最近的研究表明,NAD + 的减少是与年龄相关的代谢衰退发展的关键因素。增加NAD + 水平体内导致激活寿命和健康跨度相关因素。此外,它改善了老化的几个生理和代谢参数,包括自然和加速老化的小鼠模型中的肌肉功能,运动能力,葡萄糖耐量和心脏功能。

鉴于监测细胞NAD + 和NADH水平的重要性,有一个值得信赖的方法是至关重要的。该方案的目的是在有效且广泛适用的测定中描述来自组织和细胞的NAD + 和NADH提取以及其图形和定量分析。

【背景】氧化烟酰胺腺嘌呤二核苷酸和还原型烟酰胺腺嘌呤二核苷酸(NAD + 和NADH)是重要的生物辅助因子,它们在几种合成代谢和分解代谢功能中提供和接受电子。它们参与诸如糖酵解,三羧酸循环和氧化磷酸化的反应。此外,它还作为DNA损伤修复中涉及的几种酶的底物,例如sirtuins和poly(ADP-核糖)聚合酶(PARP)(Imai和Guarente,2014; Verdin,2015; Yoshino et al。 ,2018)。

NAD + ...

Nuclear Transformation of Chlamydomonas reinhardtii by Electroporation
Author:
Date:
2018-05-05
[Abstract]  The unicellular green alga Chlamydomonas reinhardtii is an important model organism for studying photosynthesis, acclimation to abiotic stress, cilia biology, and many other biological processes. Many molecular biology tools exist for interrogating gene function including the ability to easily transform the nuclear genome of Chlamydomonas. While technical advances such as TALENs, ZFNs and CRISPR are making it easier to precisely edit the nuclear genome, the efficiency of such methods in Chlamydomonas is at present very low. In contrast, random insertion by nuclear transformation tends to be a much more efficient process. This protocol describes a method for transformation of the Chlamydomonas nuclear genome by electroporation. The protocol requires at ... [摘要]  单细胞绿藻莱茵衣藻是研究光合作用,适应非生物胁迫,纤毛生物学和许多其他生物过程的重要模式生物。 许多分子生物学工具用于询问基因功能,包括轻松转化衣藻的核基因组的能力。 虽然TALENs,ZFNs和CRISPR等技术进步正在使精确编辑核基因组变得更加容易,但此类方法在衣原体中的效率目前非常低。 相反,通过核转变随机插入往往是一个更有效的过程。 该协议描述了通过电穿孔转化衣原体核基因组的方法。 该协议需要至少3天的工作,并通常导致1-2周内出现小菌落。

【背景】众多的分子,遗传和基因组资源使得莱茵衣藻(以下简称衣衣属)成为研究各种生物过程的优秀模式生物。已经开发了许多技术来改变衣藻核,叶绿体和线粒体,包括粒子轰击(Boynton等,1988),玻璃珠转化(Kindle,1990)和电穿孔(Shimogawara等人,1998)。核衣壳菌可通过将衣藻暴露于物理或化学诱变剂(例如紫外线或甲磺酸乙酯)而产生,但通常通过随机插入诱变转基因DNA而获得。由于衣藻核转化的同源重组效率非常低(Zorin等人,2009; Jinkerson和Jonikas,2015),转化的DNA通常整合到核基因组随机位点。存在许多用于随后鉴定异位DNA的插入位点的技术,包括经典遗传作图(Rymarquis等人,2005),TAIL-PCR(Dent等人 ...

Isolation of Genomic DNA from Chlamydomonas reinhardtii
Author:
Date:
2018-05-05
[Abstract]  Chlamydomonas reinhardtii is a soil-dwelling eukaryotic green alga that is widely used as a laboratory model organism for research on photosynthesis, ciliary biology, lipid metabolism and many other aspects of cell biology and physiology. With sequenced nuclear, chloroplast and mitochondrial genomes, Chlamydomonas is also an excellent organism for genetics and genomics research. This protocol describes the isolation of genomic DNA from Chlamydomonas using a standard phenol:chloroform extraction method followed by ethanol precipitation. The protocol requires minimal lab materials, takes approximately 4 h to complete, and can also be used for isolation of genomic DNA from other eukaryotic green algae. [摘要]  莱茵衣藻是一种土壤居住的真核绿藻,广泛用作实验室模型生物,用于光合作用,睫状生物学,脂质代谢以及细胞生物学和生理学的许多其他方面的研究。 随着核,叶绿体和线粒体基因组的测序,衣藻也是遗传学和基因组学研究的优秀生物。 该协议描述了使用标准酚:氯仿提取方法然后乙醇沉淀从衣藻(Chlamydomonas)中分离基因组DNA。 该协议需要最少的实验室材料,大约需要4小时才能完成,也可用于从其他真核绿藻中分离基因组DNA。

【背景】分离核酸是克隆和测序遗传物质的关键第一步,为从基因表达到基因进化等各种分子生物学研究提供了基础。存在许多用于从藻类中分离DNA的方案(Weeks等人,1986; Fawley和Fawley,2004; HwangBo等人,2010)。通常,通过离心沉淀细胞并在含有去污剂如SDS的缓冲液中裂解以溶解膜。随后在苯酚:氯仿中提取至少一次,并在氯仿中提取至少一次。在一些情况下,进行RNA酶处理步骤以降解RNA。然后通过加入乙醇或异丙醇沉淀DNA,并在冰上或在冰箱中孵育。沉淀并洗涤沉淀的DNA后,通常将其重悬于水或缓冲液(例如Tris-EDTA)中,并在260nm处通过分光光度法定量。

本文所述的方案利用两次苯酚/氯仿/异戊醇萃取和两次氯仿/异戊醇萃取,其间具有RNase处理步骤。在有机溶剂中加入异戊醇可防止起泡并稳定含有高浓度凝固蛋白的界面。重要的是,该协议产生高质量的基因组DNA,适用于下游应用,如克隆和测序。 ...

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