| Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
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Author:
Date:
2020-10-20
[Abstract] Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling can be accomplished using a single kinase-catalyzed phosphorylation step, existing procedures for 3′-radiolabeling often result in probe heterogeneity, prohibiting precise DNA fragment identification in downstream experiments. We present here a new protocol to efficiently attach a 32P-phosphate to the 3′ end of a DNA oligonucleotide of arbitrary sequence, relying on inexpensive DNA oligonucleotide modifications ...
[摘要] [摘要] 对DNA结合蛋白和DNA切割蛋白的生化研究通常得益于放射性标记与两个DNA末端之一的特异性连接。在许多情况下，有必要进行两种版本的同一实验：一种是5′DNA末端标记，另一种是3′DNA末端标记。虽然均匀的5′-放射性标记可以通过单个激酶催化磷酸化步骤完成，但现有的3′-放射性标记程序通常会导致探针的异质性，从而妨碍了下游实验中精确的DNA片段鉴定。我们提出了一种新的方案，利用廉价的DNA寡核苷酸修饰（2′-O-甲基核糖核酸和核糖核酸糖取代）、两种酶（T4多核苷酸激酶和T4 RNA连接酶2），将32P磷酸有效地连接到任意序列的DNA寡核苷酸的3′端，以及DNA和RNA对氢氧化物处理的差异敏感性。该方法制备的放射性探针分子具有均一性和氧化剂相容性，可用于DNA酶特性和DNA足迹分析中的精确切割位点定位。
[背景] ...
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| Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
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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 / ...
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| Mismatched Primer Extension Assays
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Author:
Date:
2015-06-20
[Abstract] Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg2+ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg2+ ...
[摘要] 稳态动力学测定法是估计几种聚合酶的保真度的可靠方法（Menendez-Arias，2009; Rezende和Prasad，2004; Svarovskaia等人，2003）。分析具有在3'末端的特异性错配的引物的延伸的能力是错配引物延伸测定的主要强度。最近，我们使用错配的引物延伸测定法显示当Mg 2+浓度降低到生理相关浓度（〜0.25mM）时，HIV RT的保真度显着增加（Achuthan等al 。，2014）。在这里，我们详细地描述如何进行错配引物延伸测定以使用人类免疫缺陷病毒逆转录酶（HIV RT）在2mM Mg 2+ 2 + 测量标准延伸效率。然后可以使用标准延伸效率估计聚合酶的相对保真度。这里描述的测定基于在Mendelman等人（1990）中公开的方法。
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