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Company: New England Biolabs
Catalog#: R3535L
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In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
[Abstract]  The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their ‘correct’ strands, as well as quality control mechanisms that evict polymerases when they associate with an ‘incorrect’ strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins. [摘要]  真核生物复制品是重复DNA的多蛋白复合物。 复制品被雕刻成连续的前导链合成与不连续的滞后链合成,主要通过DNA聚合酶ε和δ以及解旋酶,聚合酶α-引发酶,DNA滑动夹,夹带载体和许多其它蛋白质进行。 我们以前已经建立了聚合酶ε和δ靶向其“正确”链的机制,以及在与“不正确”链相关联时驱赶聚合酶的质量控制机制。 在这里,我们提供了使用纯蛋白质在体外差异测定前导和滞后链复制的实用指南。
Using pure proteins from Saccharomyces cerevisiae, our lab was the first to reconstitute a functional eukaryotic DNA replisome, a ~2 MDa complex that includes the 11-subunit CMG helicase (complex of Cdc45, Mcm2-7, GINS heterotetramer), the 4-subunit DNA polymerase (Pol) ε, the 4-subunit Pol α-primase, the PCNA (Proliferating Cell Nuclear Antigen) clamp homotrimer ring shaped processivity factor that ...

CRISPR/Cas9 Editing of the Bacillus subtilis Genome
[Abstract]  A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with unparalleled facility. CRISPR/Cas9 has allowed for genome edits to be more precise, while also increasing the efficiency of transferring mutations into a variety of genetic backgrounds. As a result, the advantages are realized in tractable organisms and organisms that have been refractory to genetic manipulation. Here, we describe our method for editing the genome of the bacterium Bacillus subtilis. Our method is highly efficient, resulting in ... [摘要]  大多数现代生物学家的基本过程是研究生物体的遗传操作。尽管许多不同的方法用于编辑细菌基因组已经在实验室中使用了数十年,但CRISPR / Cas9技术对细菌遗传学的适应使得研究人员能够以无与伦比的设施来操纵细菌基因组。 CRISPR / Cas9允许基因组编辑更精确,同时也提高将突变转移到各种遗传背景的效率。因此,在遗传操作难以处理的易处理生物和生物体中实现了这些优点。在这里,我们描述了我们编辑枯草芽孢杆菌细菌基因组的方法。我们的方法是高效的,导致精确,无标记的突变。此外,在产生编辑质粒之后,可以将突变快速导入几个遗传背景,大大增加可进行遗传分析的速度。

枯草芽孢杆菌是高度易处理的革兰氏阳性菌。遗传研究适用于使用多种载体通过同源重组快速有效地引入突变。尽管有许多不同的方法来引入B突变。 subtilis,每种方法都有其局限性。一种简单而简单的方法,用于在B中进行突变。枯草芽孢杆菌是基因破坏,其中将质粒整合到感兴趣的基因内(Vagner等人,1998)。主要的局限性包括:1)扰乱操纵子的极地作用的潜力; 2)引进和保留外来DNA; 3)一旦使用抗生素耐药性盒,如果在其他突变的背景下研究给定的突变,则研究者必须使用不同的盒;和4)该方法限于靶向整个基因,并且不能产生更精确的点突变。 ...