Plasmid Extract from Budding Yeast (Saccharomyces cerevisiae)
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Author:
Date:
2018-07-20
[Abstract] Plasmids are widely used tools in yeast research. In many cases, plasmid libraries are used in genetic screens or in yeast two hybrid screens. In such cases, it is necessary to extract plasmids carrying unknown genetic elements from positive clones that were isolated in the screen.
This is a simple protocol to extract plasmid DNA from budding yeast cultures (Robzyk and Kassir, 1992). The amount produced is small, but it is sufficient for PCR or for transformation into bacteria, where the plasmid can be amplified to provide sufficient amounts for downstream uses (e.g., restriction enzyme analysis, sequencing).
[摘要] 质粒是酵母研究中广泛使用的工具。 在许多情况下,质粒文库用于遗传筛选或酵母双杂交筛选。 在这种情况下,有必要从筛选中分离的阳性克隆中提取携带未知遗传元件的质粒。
这是从芽殖酵母培养物中提取质粒DNA的简单方案(Robzyk和Kassir,1992)。 产生的量很小,但它足以用于PCR或转化到细菌中,其中质粒可以被扩增以提供足够的量用于下游用途(例如,,限制酶分析,测序)。
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Method for CRISPR/Cas9 Mutagenesis in Candida albicans
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Author:
Date:
2018-04-20
[Abstract] Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that allow simple ligation of synthetic duplex 23mer guide oligodeoxynucleotides for high copy gRNA expression in C. albicans strains that express codon-optimized Cas9. This protocol allows isolation and characterization of deletion strains within nine days.
[摘要] 白色念珠菌是最普遍和最重要的人类真菌病原体。 CRISPR作为基因编辑手段的出现极大地促进了 C中的遗传分析。白色假丝酵母。 在这里,我们描述一个详细的分步过程来构建和分析 C。 白色念珠菌缺失突变体。 该协议使用质粒,允许合成的双链体23mer引导寡聚脱氧核苷酸在高拷贝gRNA表达的简单连接。 表达密码子优化的Cas9的白色念珠菌菌株。 该协议允许在9天内分离和鉴定缺失菌株。
【背景】℃。白色念珠菌是一种难以处理遗传的有机体。由于它通常作为不容易进行有性生殖的二倍体存在,所以纯合隐性突变需要对每个基因座进行连续修饰。克隆间规则间隔短回文重复(CRISPR)突变的发展和应用。白色念珠菌促进遗传操作,因为它允许两个等位基因同时突变(Vyas et al。,2015; Min et al。,2016; Ng and Dean,2017 )。 CRISPR基因编辑涉及将RNA引导的核酸酶募集至邻近NGG原型间隔子邻接基序(PAM)的互补靶位点(Jinek等人,2012; Cong等人, 2013年;马里等人,2013年)。 CRISPR相关(Cas)核酸酶通过与结合Cas9的激活CRISPR RNA(tracrRNA)相关的指导RNA之间的互补碱基配对以高特异性进行靶向(Gasiunas等人, ...
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Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
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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 creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining ...
[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; ...
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