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QIAQuick PCR Clean-up Kit

QIAquick PCR纯化试剂盒

Company: QIAGEN
Catalog#: 28104
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Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy
Author:
Date:
2021-03-05
[Abstract]  

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to

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[摘要]  [摘要]在细菌中,停滞的DNA复制叉的重新启动需要DN A解旋酶PriA 。PriA可以识别并重塑废弃的DNA复制叉,在3'到5'方向展开DNA,并促进解旋酶DnaB加载到DNA上以重新开始复制​​。ssDNA结合蛋白(SSB)通常存在于废弃的叉子上,从而保护ssDNA免受核酸酶的破坏。该研究是基于所述测定法离解结,表面等离振子共振,单分子FRET,和x射线晶体结构已经揭示的解旋酶活性PRIA ,SSB- PriA相互作用以及PriA解旋酶的结构信息。在这里,我们使用原子力显微镜(AFM)可视化了在不存在ATP的情况下在有或没有SSB的情况下PriA和带有或不带有SSB的DNA底物之间的相互作用,以描绘PriA在其ATP催化的DNA解链反应之前的底物识别模式。该协议描述了获取高分辨率AFM图像的步骤以及数据分析和表示的细节。

[背景]当DNA复制遇到障碍或断裂时,需要对其进行修复并随后重新启动(Kogoma,1997; Cox等,2000; McGlynn和Lloyd,2002;G abbai和Marians,2010; Michel等,2018)。 )。在细菌中,DNA解旋酶PRIA通过识别废弃DNA复制叉,从而便于重新组装的介导这一过程复制体的解旋酶和装载DNAB (Wickner和赫维茨,1975; Zavitz和Marians,1992; ...

Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes
Author:
Date:
2021-03-05
[Abstract]  

Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant library in traditional TIS, the defined mutant library sequencing (DML-Seq) has advantages as: 1) efficient mutagenesis; 2) low bottleneck effects; 3) avoid hotpots caused by screening; 4) can be directly used in the following experiments. Here, we described an optimized procedure of DML-Seq for fitness screen to supply classical TIS using the marine pathogenic bacterium Edwardsiella piscicida as an example.

[摘要]  [摘要]转座子插入测序(TIS)是一项新兴技术,它利用大量的转座子突变体文库筛选特定表型,并结合高通量并行测序技术,在不同条件下确定细菌适应性的条件性基本遗传要求。与传统TIS中的大规模突变文库相比,已定义的突变文库测序(DML-Seq)具有以下优势:1)高效诱变;2)瓶颈效应低;3)避免因筛选引起的火锅;4)可直接用于以下实验。在这里,我们描述DML-SEQ的优化过程进行健身屏幕使用海洋致病菌提供古典TIS爱德华piscicida作为一个例子。


[背景]转座子插入诱变与下一代测序(NGS)结合已被证明是在多种条件下研究基因功能的有效方法(Chao等,2016; Price等,2018)。通常,TIS分析由转座子插入位点的大规模平行测序和大量插入事件的统计分析组成。

基于TIS的筛选可以在多种条件下基于高度饱和的转座子突变体文库的细菌适应性,提供单个基因座和域的适应性贡献的高分辨率图(Chao等,2016)。每个位点的插入频率或相应突变体的相对丰度通常与施加选择性压力(例如宿主和抗生素施加的压力)后与基因座对适应性的贡献成反比(Chao等人,2016)。这种方法的原理是多种相关方法的基础,包括TIS,转座子测序(TnSeq ),插入测序(INSeq ),转座子定向插入位点测序(TraDIS ...

Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System
Author:
Date:
2021-01-20
[Abstract]  

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear

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[摘要]  [摘要] 免疫耐受和应答都很大程度上由抗原呈递细胞(APC)表达的主要组织相容性复合物(MHC),T细胞上的T细胞受体(TCR)及其同源抗原之间的相互作用驱动。相互作用障碍导致自身免疫性疾病(例如1型糖尿病)的发病机理。因此,鉴定自身反应性T细胞的抗原表位导致治疗和生物标志物的重要进展。下一代测序方法可从单个T细胞快速鉴定数千种TCR克隆型,因此需要确定已鉴定TCR的同源抗原。该协议描述了T细胞活化的报告系统,其中荧光报告蛋白ZsGreen-1由活化T细胞的核因子(NFAT)信号驱动并通过流式细胞仪读取。记者T细胞也组成性表达额外的一对荧光素tein作为识别物,允许同时多路复用多达8种不同的报告T细胞系,每种表达不同的目标TCR,可通过流式细胞仪区分。一旦制成TCR表达细胞系,仅需一个转导步骤即可将其无限期用于制备新的T细胞系。这种多路复用系统允许筛选TCR-抗原相互作用的数量,否则这些相互作用将是不切实际的,可在多种情况下使用(即,筛选单个抗原或抗原库),并可用于研究任何T细胞-MHC-抗原三分子相互作用。

[背景] T细胞,抗原呈递细胞(APC)及其同源抗原之间的相互作用是自身免疫性疾病(例如1型糖尿病)的主要事件(Michels等,2017; ...

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