{{'Search' | translate}}
 

Manganese(II) chloride tetrahydrate

氯化锰(II)四水合物

Company: Sigma-Aldrich
Catalog#: M3634
Bio-protocol()
Company-protocol()
Other protocol()

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。 ...

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; ...

Comments