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Vortex-Genie 2

Company: Dutscher
Catalog#: Vortex-Genie 2
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Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap
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Date:
2018-10-20
[Abstract]  This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap. [摘要]  该协议描述了CRISPR-Trap从设计基因靶向策略到验证成功编辑的克隆的应用,所述克隆在各种人细胞系上得到验证,其中人类诱导的多能干细胞(hiPSC)。 CRISPR-Trap优于常规方法的优点是从靶基因完全去除任何内源全长转录物。 CRISPR-Trap适用于在其第一个外显子中没有编码序列或编码序列很少的任何靶基因。 到目前为止,已经使用CRISPR-Trap成功编辑了几种人细胞系和不同基因。

【背景】CRISPR / Cas9技术的出现促进了基因敲除和基因编辑的基因组靶向。执行敲除的常规方法依赖于引入移码导致过早终止密码子(PTC),截短开放阅读框(ORF)以及随后通过无义介导的mRNA衰变(NMD)降解靶基因的转录物。 。这种方法的一个可能的缺陷是全长转录物,其可以逃避NMD并产生具有残余或甚至显性负功能的C末端截短蛋白。该协议提出了CRISPR-Trap,这是我们最近建立的一种方法(Reber et al。>,2018),成功编辑后将阻止从靶基因位点表达任何全长转录本(图1)。简而言之,这种方法针对CRISPR / ...

Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
Author:
Date:
2018-10-05
[Abstract]  Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ... [摘要]  核糖核蛋白颗粒(mRNP)是由mRNA和RNA结合蛋白(RBP)组成的复合物,其控制mRNA转录定位,转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件,如poly(A)尾或5'帽结构,这通过应用广泛使用的方法如oligo(dT)纯化来阻止它们的鉴定。在这里,我们描述了基于现有的交联和免疫沉淀(CLIP)方法的修饰的交联亲和纯化方案(cCLAP),以分离mRNP中可被去腺苷酸化,去除和/或部分降解的mRNA,从而开启了检测不同的可能性。非编码RNA(ncRNA)的类型。分离后,将RNA进行衔接子连接,然后进行下一代测序(NGS)。由于快速有效的交联和淬灭步骤,该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体(PB)或应力颗粒(SG)。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀,然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法(Tagwerker et al。,2006; Hafner et al。,2010; Kishore et al。,2011)。 ...

Real-time PCR Analysis of PAMP-induced Marker Gene Expression in Nicotiana benthamiana
Author:
Date:
2018-10-05
[Abstract]  Perception of pathogen-associated molecular patterns (PAMPs) often triggers various innate immune responses in plants. The transcriptional changes of defense-related genes are often used as a marker to assay PAMP-triggered plant immune response. Here we described a protocol to monitor the relative expression level of marker genes in Nicotiana benthamiana upon treatment with PAMPs. The procedure includes leaf treatment using PAMPs, total RNA isolation, cDNA synthesis, quantitative real-time PCR and data analysis. This protocol is applicable to monitor marker gene expression triggered by different PAMPs in N. benthamiana. [摘要]  对病原体相关分子模式(PAMP)的感知经常引发植物中的各种先天免疫应答。 防御相关基因的转录变化通常用作测定PAMP触发的植物免疫应答的标记。 在这里,我们描述了一种方案,用于监测用PAMP处理的本塞姆氏烟草中的标记基因的相对表达水平。 该方法包括使用PAMP进行叶处理,总RNA分离,cDNA合成,定量实时PCR和数据分析。 该协议适用于监测 N中不同PAMP触发的标记基因表达。本塞姆氏。
【背景】病原体相关的分子模式,即PAMP,是一类源自病原体的分子,在微生物中相对保守。多个PAMP,如flg22和XEG1(Felix et al。,1999; Ma et al。,2015),已被表征,可通过植物细胞表面定位模式检测 - 识别受体(PRR),从而诱导PAMP引发的免疫(Couto和Zipfel,2016)。 PAMP触发的主要反应之一是与防御相关的制造者基因的激活(Navarro et al。,2004; Zipfel et al。,2006)。 Nicotiana benthamiana 已被广泛用作模型植物,并且对多种PAMP敏感。在 N.宾夕法尼亚,先前发现了标记基因,如 NbCYP71D20 , NbACRE31 和 NbWRKY22 ,它们在PAMP处理后迅速活化( Heese et al。,2007; Segonzac et ...

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