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UltraPureTM Agarose

UltraPure TM琼脂糖

公司名称: Thermo Fisher Scientific
产品编号: 16500500
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Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
Author:
Date:
2018-08-05
[Abstract]  Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments ... [摘要]  染色体构象捕获测序(Hi-C)是一种全面询问细胞核中染色质三维定位的有效方法。 Hi-C的发展可以追溯到染色体构象捕获的分辨率和通量的连续增加(3C)(Dekker et al。,2002)。 3C的基本工作流程包括(i)通常用甲醛固定完整的染色质,(ii)用限制酶切割固定的染色质,(iii)在稀释条件下重新连接粘性末端,以促进交联片段之间的连接或随机片段之间的那些和(iv)量化基因组基因座对之间的连接事件的数量(de Wit和de Laat,2012)。在最初的3C方案中,通过半定量PCR扩增对应于少量基因组位点(“一对一”)的选定连接接头来测量连接频率(Dekker et al。,2002 )。然后,染色体构象捕获芯片(4C)和染色体构象捕获碳复制(5C)技术扩展3C以分别以“一对多”或“多对多”方式计算结扎事件。 Hi-C(Lieberman-Aiden et al。,2009)最终将3C与下一代测序相结合(Metzker,2010)。此处,在再连接之前,用生物素标记的核苷酸类似物填充粘性末端以在后续步骤中富集具有连接连接的片段。然后对Hi-C文库进行高通量测序,并将得到的读数映射到参考基因组,允许以“多对多”方式确定接触概率,其分辨率仅受限制性位点的分布限制和阅读深度。 Hi-C的首次应用是阐明人类基因组中的全球染色质折叠原理(Lieberman-Aiden et ...

Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
Author:
Date:
2018-03-20
[Abstract]  Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease ... [摘要]  芽殖酵母中的基因组修饰已经非常成功,主要归功于其高度同源性的DNA修复机制。之前已经描述了几种用于修饰酵母基因组的方法,其中许多方法涉及至少两个步骤:插入选择标记并用该标记取代预期的修饰。在这里,我们描述了CRISPR-Cas9介导的基因组编辑方案,用于在没有任何选择标记的情况下在单步转化中修饰任何感兴趣的酵母基因(基本或非必需)。在该系统中,Cas9核酸酶在选择的基因座处产生双链断裂,这在酵母细胞中通常是致死的,而不管由于无效的非同源末端连接修复导致的靶基因座的重要性。该致死性通过使用源自PCR的修复模板的同源重组导致有效的修复。在涉及必需基因的情况下,用功能性等位基因编辑基因组病变的必要性作为额外的选择层。作为一个激励性的例子,我们描述了使用这种策略替代HEM2,一种必需的酵母基因,以及相应的人类直向同源物ALAD。

【背景】酿酒酵母(Baccharomyces cerevisiae,Baker's酵母)作为一种遗传易处理的生物体具有悠久的历史,并且有许多操作酵母基因组的方法。然而,直到最近,有必要应用选择以分离具有所需遗传改变的克隆(Kearse等人,2012; DiCarlo等人,2013; Lee等人,等,2015; ...

Detection and Analysis of Circular RNAs by RT-PCR
Author:
Date:
2018-03-20
[Abstract]  Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) ... [摘要]  真核细胞中的基因表达在转录和转录后水平受到严格调控。 mRNA转录,mRNA转录和mRNA翻译等后转录过程由RNA结合蛋白(RBPs)和非编码(nc)RNAs控制。大量的ncRNA家族包含多种调控RNA,如microRNAs和长的非编码(lnc)RNAs,但也是探索不足的一类环状RNAs。虽然三十多年前电子显微镜首次发现,但只有高通量RNA测序(RNA-seq)的出现和创新生物信息学管道的开发已经开始允许系统鉴定circRNA(Szabo和Salzman,2016;熊猫,2017b;熊猫等,2017c)。然而,通过RNA测序鉴定的真正的circRNA的验证需要其他分子生物学技术,包括常规或定量(q)聚合酶链反应(PCR)和Northern印迹分析(Jeck和Sharpless,2014)的逆转录(RT)。使用不同引物的环状RNA的RT-qPCR分析已被广泛用于检测,验证和有时定量circRNA(Abdelmohsen等人,2015和2017; Panda等人, ,2017b)。如在此详述的,设计为跨越循环RNA后接连接序列的分歧引物可以特异性扩增circRNA而不是对应的线性RNA。总之,使用不同引物的RT-PCR分析允许直接检测和定量circRNA。

【背景】CircRNAs是共价闭合的,缺少5'或3'末端的单链RNA。虽然它们的起源知之甚少,但它们可以通过称为反向剪接的过程从前体mRNA产生(Panda等人,2017d; ...

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