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Axygen® 0.2 mL Thin Wall PCR Tubes with Flat Cap

0.2mL带平盖的薄壁PCR管

Company: Corning
Catalog#: PCR-02-C
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Brain Tissue Culture of Per2::Luciferase Transgenic Mice for ex vivo Bioluminescence
Author:
Date:
2018-07-05
[Abstract]  In circadian research, it is essential to be able to track a biological rhythm for several days with the minimum perturbation for the organisms or tissues. The use of transgenic mice lines, in which the luciferase reporter is coupled to a molecular clock protein (here PERIOD2), gives us the opportunity to follow the circadian activity in different tissues or even single clock cells for days without manipulation. This method creates sections using a mouse brain matrix, which allows us to obtain several brain samples quickly at a single time point. [摘要]  在昼夜节律研究中,能够以最小的生物或组织扰动跟踪生物节律数天是至关重要的。 使用转基因小鼠系,其中荧光素酶报告基因与分子钟蛋白(此处为PERIOD2)偶联,使我们有机会在不经操作的情况下跟踪不同组织或甚至单个时钟细胞中的昼夜节律活动数天。 该方法使用鼠标脑矩阵创建切片,这允许我们在单个时间点快速获得几个脑样本。

【背景】昼夜节律是大约24小时循环的行为或分子变化,并且在没有任何外部线索的情况下持续。在哺乳动物中,运动活动,体温和激素释放是昼夜节律的实例,其在位于下丘脑的视交叉上核(SCN)时钟的控制下。 SCN细胞保持内源性节律的能力是由于时钟基因表达的正负循环组成的分子机制:首先,CLOCK和BMAL1蛋白异二聚化通过E-box位点激活不同基因的转录关于基因如 period ( Per1-3 )和隐花色素( Cry1-2 ; Takahashi)的启动子 et al。,2008)。然后,PERIOD和CRYPTOCHROME的蛋白质异二聚化并返回到细胞核以防止BMAL1与E-Box结合。因此,PERIOD和CRYPTOCHROME抑制其自身的转录(Takahashi et al。,2008)。第二个环由类视黄醇相关的孤儿受体(ROR)和Rev-Erb组成:ROR蛋白激活 Bmal1 基因,而REV-ERB蛋白抑制它通过 ROR反应 Bmal1 ...

Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases
Author:
Date:
2018-02-05
[Abstract]  Protein tagging is a powerful tool for performing comprehensive analyses of the biological functions of a protein of interest owing to the existence of a wide variety of tags. It becomes indispensable in some cases, such as in tracking protein dynamics in a live cell or adding a peptide epitope due to the lack of optimal antibodies. However, efficiently integrating an array of tags into the gene of interest remains a challenge. Traditional DNA recombinant technology based on type II restriction endonucleases renders protein tagging tedious and inefficient as well as the introduction of an unwanted junction sequence. In our attempt to tag Thrombospondin type 1 domain-containing 1 (THSD1) that we identified as the first intracranial aneurysm gene (Santiago-Sim et al., 2016), we ... [摘要]  由于各种标签的存在,蛋白质标签是一种对感兴趣的蛋白质的生物学功能进行全面分析的有力工具。在某些情况下,例如跟踪活细胞中的蛋白质动态变化或由于缺乏最佳抗体而添加肽表位,这变得不可或缺。然而,将一系列标签有效地整合到感兴趣的基因中仍然是一个挑战。基于II型限制性内切核酸酶的传统DNA重组技术使得蛋白质标记繁琐且效率低下以及引入不需要的连接序列。我们试图标记我们确定为第一个颅内动脉瘤基因的血小板反应蛋白1型结构域1(THSD1)(Santiago-Sim等人,2016),我们开发了一种新型精确标记技术,组合使用II型和IIS限制性核酸内切酶(Xu等人,2017),其产生高效率的无缝克隆。在这里,我们描述了一个协议,不仅为任何感兴趣的基因提供了一个广义的策略,而且还将THSD1中的11个不同标签的应用作为一个循序渐进的例子。


【背景】具有不同特征的多功能标签可以作为一组分析蛋白质功能的工具。诸如绿色荧光蛋白(GFP)标签及其衍生物,串联亲和纯化标签(如FLAG-HA或ProtA-CBP)的各种标签多年来革新了生物学研究。一些新开发的化学标签,如SNAP或CLIP,允许以时间控制的方式有条件地标记感兴趣的蛋白质(Bodor等人,2012)。然而,有效地将尽可能多的不同标签整合到感兴趣的基因中的方法发展不足。

传统的DNA重组利用识别回文序列的II型限制性内切核酸酶。例如,Eco ...

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol
Author:
Date:
2016-12-20
[Abstract]  The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

Here, we present a protocol for single-step marker switching by ...
[摘要]  利用不同的选择标记来标记或突变粟酒裂殖酵母中的多个基因的能力受到仅仅两种可选择标记的历史使用的阻碍,即 +和 kanMX6 ;后者赋予抗生素G418(遗传霉素)抗性。最近已经描述了更多的标记物,但是将它们引入酵母细胞通常需要从头开始施加应变。为了克服这个问题,我们和其他团队已经创建了具有与 + 和 kanMX6 的侧翼同源性的转换盒,这使得能够有效和省时的方式在现有的突变或标记的裂变酵母菌株中交换标记。
 在这里,我们提出了裂殖酵母,粟酒裂殖酵母(Schizosaccharomyces pombe)中醋酸锌转化的单步标记转换方案。以下我们将介绍如何将 ura4 + 标记交换到kanMX6 , natMX4 或 hphMX4标记,其分别对抗生素G418,海参(clonNAT)或潮霉素B提供抗性。我们还详细介绍了如何交换营养标记的任何 MX 标记,例如 arg3 + , his3 + , leu1 + 和 ura4 + 。

背景 这种用于粟酒裂殖酵母的单步骤标记交换方案允许将标记有类型的抗生素标记的任何标记或突变的基因替换为营养标记物(含有arg3 + , + ,并且已经构建了 ura4 + )并且为任何 MX交换遗传 ura4 + ...

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