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Sodium dodecyl sulfate

十二烷基硫酸钠

Company: Sigma-Aldrich
Catalog#: L5750
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RNA Immunoprecipitation (RIP) Sequencing of Pri-miRNAs Associated with the Dicing Complex in Arabidopsis
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Date:
2018-07-05
[Abstract]  RNA immunoprecipitation (RIP) is an antibody-based technique used to map in vivo RNA-protein interactions. DBR1, an RNA debranching enzyme, is responsible for the debranching of lariat RNA, for the degradation and turnover of lariat RNAs. It is well known that primary miRNA (Pri-miRNA) is recognized and further processed into mature miRNA by the Dicing complex mainly composed of DCL1 and HYL1. Due to the low abundance of pri-miRNAs, RIP followed qRT-PCR has been widely used to evaluate the binding efficiency of the Dicing complex with pri-miRNAs in previous studies. Therefore, the genome-wide evaluation of the Dicing complex with pri-miRNAs is lacking. With the improvement of high-throughput sequencing technologies, we successfully used RIP-seq to compare the binding efficiency ... [摘要]  RNA免疫沉淀(RIP)是一种基于抗体的技术,用于绘制体内 RNA-蛋白质相互作用。 DBR1是一种RNA脱支酶,负责套索RNA的脱支,用于套索RNA的降解和转换。众所周知,主要miRNA(Pri-miRNA)被主要由DCL1和HYL1组成的切割复合物识别并进一步加工成成熟miRNA。由于pri-miRNA的丰度较低,RIP随后的qRT-PCR已被广泛用于评估切割复合物与pri-miRNA在先前研究中的结合效率。因此,缺乏对具有pri-miRNA的切割复合物的全基因组评估。随着高通量测序技术的改进,我们成功地使用RIP-seq比较了Dicing复合物与野生型和 dbr1-2 突变体之间的pri-miRNA的结合效率。 。在该方案中,我们提供了在两种不同基因型之间的HYL1-YFP和DCL1-YFP转基因植物中使用GFP捕获珠的RIP-seq的详细描述。该方法可用于评估pri-miRNA与拟南芥中的切割复合物的结合,并且它可以应用于植物中的其他RNA结合蛋白。

Nuclear Transformation of Chlamydomonas reinhardtii by Electroporation
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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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