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T4 DNA Ligase

T4 DNA连接酶

Company: New England Biolabs
Catalog#: M0202S
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Reduced Representation Bisulfite Sequencing in Maize
Author:
Date:
2018-03-20
[Abstract]  DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies (Smith et al., 2009). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore ... [摘要]  DNA甲基化是调节植物发育的表观遗传修饰(Law and Jacobsen,2010)。全基因组亚硫酸氢盐测序(WGBS)是用单碱基分辨率分析全基因组甲基化模式的最先进的方法(Cokus et al。,2008)。然而,对于具有大基因组的生物体,例如玉米的2.1Gb基因组,WGBS可能非常昂贵。代表性亚硫酸氢盐测序(RRBS)已经在哺乳动物研究中发展(Smith等人,2009)。通过用大小选择范围大约40-220bp的 Msp 消化基因组,可以对仅涵盖〜1%人类基因组的CG富含区域进行特异性测序。然而,与哺乳动物基因组不同,植物基因组不显示清楚的CpG岛。因此原来的RRBS协议不适用于工厂。因此,我们开发了一种计算机管道来选择特定的酶以生成感兴趣区域(ROI) - 富集的,例如,富含启动子的,减少的植物表达基因组(例如, Hsu et al。,2017)。通过用MseI消化玉米基因组并选择40-300bp片段,我们测序了大约四分之一的玉米基因组,同时保留了84.3%的启动子信息。该协议已在玉米中成功建立,可广泛应用于任何基因组。我们的计算机管道系统与RRBS文库制备方案相结合,允许进行计算分析和实验验证。

【背景】DNA甲基化是一种可遗传的表观遗传修饰,通过调节基因表达和染色质结构在动物,植物和真菌的许多发育过程中发挥重要作用(Law and ...

Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
Author:
Date:
2018-01-20
[Abstract]  With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the extensively studied Gram-negative bacterium Escherichia coli, as it is considered as the workhorse for both research and industrial purposes. Here, we present a simple, robust and effective protocol using the CRISPR/Cas9 system in combination with the λ Red machinery for gene knockout in E. coli. Crucial in our procedure is the use of a double-stranded donor DNA and a curing strategy for removal of the guide RNA encoding plasmid ... [摘要]  随着CRISPR / Cas9技术作为基因组编辑的标准工具的最近实施,全世界的实验室正在经历PCR以来分子生物学方面最大的进步之一。这种方法的关键优点是其简单和普遍适用于任何物种的门。特别感兴趣的是广泛研究的革兰氏阴性细菌大肠杆菌,因为它被认为是研究和工业用途的主力。在这里,我们提出了一个简单,强大和有效的协议,使用CRISPR / Cas9系统结合λ红色基因敲除机器。大肠杆菌。在我们的程序中最重要的是使用双链供体DNA和固化策略来去除导向RNA编码质粒,其允许在仅仅两个工作日后开始新的突变。我们的方案允许多个具有高诱变效率的基因敲除株,适用于高通量的方法。

【背景】革兰氏阴性细菌大肠杆菌是生物技术工程中最重要的生物之一。已在能源,农业,食品生产,生物技术,医药等不同行业的各种流程中成功实施。由于不断的技术进步,生物技术部门正在迅速发展。特别是,CRISPR / Cas9技术可能是PCR(分子)生物学最大的革命(Ledford,2015)。简而言之,CRISPR / Cas9保护细菌免受诸如质粒和病毒等侵入性遗传因子的影响(Marraffini,2015)。利用这种从原核生物获得的免疫系统,已经开发了基于CRISPR / Cas9系统的基因组编辑的非常有力的工具(Jinek等人,2012)。

CRISPR / ...

Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System
Author:
Date:
2017-08-05
[Abstract]  We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages. This protocol allows seamless deletion of desired genes in a T7 phage, and can be expanded to other phages and other types of genetic manipulations as well. [摘要]  我们提出了一种用于从T7噬菌体基因组中删除基因的基于CRISPR-Cas的技术。 首先将编码与待缺失的靶基因的同源臂的DNA片段克隆到质粒中。 然后将T7噬菌体在携带该质粒的大肠杆菌中繁殖。 在这种繁殖期间,一些噬菌体基因组与质粒进行同源重组,从而缺失靶基因。 为了选择这些基因组,CRISPR-Cas系统用于切割未编辑的基因组,从而能够分离所需的重组噬菌体。 该协议允许在T7噬菌体中无缝地删除所需的基因,并且可以扩展到其它噬菌体和其他类型的遗传操作。
【背景】噬菌体(噬菌体)是生物圈中最普遍和广泛分布的生物实体,突出了它们的生态重要性(Suttle,2007)。许多研究还提出将噬菌体用于医疗目的(Weber-Dabrowska等人,2001; Merril等人,2003; Harper和Enright,2011; Edgar ,2012; Bikard等人,2014; Citorik等人,2014; Yosef等人, 2014年和2015年)。不幸的是,仅有少数公开的方法详细描述了噬菌体基因组学的基因工程(Selick等人,1988; Marinelli等人,2008; ...

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