| 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., ...
[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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| 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 ...
[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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| Metabolic Labeling of Yeast RNA with Radioactive Uracil
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Author:
Date:
2013-04-05
[Abstract] To examine gene expression, Northern blot or Real-Time PCR can be used to detect low abundant RNA such as mRNA. However, for high abundant RNAs such as rRNA and tRNA, Northern blot will not be able to discriminate the newly synthesized RNA from total RNA. Therefore, metabolic labeling is necessary to evaluate the expression of rRNA and tRNA genes. In this protocol, I describe a step-by-step method for labeling yeast RNA with radioactive uracil and examine the synthesis of these high abundant RNAs.
[摘要] 为了检查基因表达，可以使用Northern印迹或实时PCR来检测低丰度RNA如mRNA。 然而，对于高丰度的RNA如rRNA和tRNA，Northern印迹将不能区分新合成的RNA与总RNA。 因此，代谢标记是必要的，以评估rRNA和tRNA基因的表达。 在这个协议，我描述了一个分步的方法，用放射性尿嘧啶标记酵母RNA，并检查这些高丰度RNA的合成。
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