C1000 Touch^TM Thermal Cycler

Company: Bio-Rad Laboratories

Catalog#: C1000 Touch^TM

Revised iCLIP-seq Protocol for Profiling RNA–protein Interaction Sites at Individual Nucleotide Resolution in Living Cells

Syed Nabeel-Shah and Jack F. Greenblatt

Published online: 2023-06-05

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Microtitre Plate Based Cell-SELEX Method

Author:

Munish Shorie and Harmanjit Kaur,

Date:

2018-10-20

[Abstract] Aptamers have emerged as a novel category in the field of bioreceptors due to their wide applications ranging from biosensing to therapeutics. Several variations of their screening process, called SELEX have been reported which can yield sequences with desired properties needed for their final use. We report a facile microtiter plate-based Cell-SELEX method for a gram-negative bacteria E. coli. The optimized protocol allows the reduction of number of rounds for SELEX by offering higher surface area and longer retention times. In addition, this protocol can be modified for other prokaryotic and eukaryotic cells, and glycan moieties as target for generation of high affinity bio-receptors in a short course of time in-vitro. [摘要] 由于适体的广泛应用，从生物传感到治疗，适体已经成为生物受体领域的一个新类别。已经报道了它们的筛选过程的几种变体，称为SELEX，其可以产生具有最终使用所需的所需性质的序列。我们报告了一种基于微量滴定板的Cell-SELEX方法，用于革兰氏阴性菌 E.大肠杆菌>。优化的协议允许通过提供更高的表面积和更长的保留时间来减少SELEX的轮数。此外，该方案可以针对其他原核和真核细胞进行修饰，并且聚糖部分可以作为在短时间内体外产生高亲和力生物受体的靶标>。

【背景】适体是1990年描述的单链合成DNA或RNA（Ellington和Szostak，1990; Tuerk和Gold，1990），具有独特的3D几何结构，这是其序列的表现。不同的序列允许合成适体，其可以结合从小分子到大蛋白质的分子阵列。这使得适配体成为常规抗体的竞争对手，常规抗体由于结构限制而限于蛋白质作为其靶标，并且不能针对高风险病原体产生，因为这些通常比产生高亲和力所需的时间更早地杀死宿主。抗体。适体在体外>设置中产生，因此可以有效地用于高风险病原体。 SELEX（ S L > > > > > > > > >分离导致在低nM范围内具有解离常数的池的筛选。已经提出了SELEX的几种变体，其使用各种固定基质，包括毛细管电泳SELEX，基于亲和层析的SELEX，磁珠SELEX，体内> ...

Quantifying Gene Expression Directly from FACS Using Hydrolysis (TaqMan) Probes on Flash-frozen Cells

Author:

Kirsten A. Copren, Oyang Teng, Cassandra J. Adams, Cláudia Bispo and Michael Lee,

Date:

2018-04-20

[Abstract] The TaqMan Gene Expression Cells-to-Ct^TM Kit enables reverse transcription directly from cell lysates without the need for isolating RNA. The recommended input range for the lysis reaction is between 10-10⁵ cells, though the upper limit may vary somewhat according to cell type. This protocol is partially adapted from the manufacturer’s protocol (TaqMan Gene Expression Cells-to-Ct^TM Kit User Guide) for gene expression for use with frozen cell pellets to allow for sample transfer among laboratories and involves experimentally confirmed dilution prior to lysis. Within this constraint, the recommended range for sample cell number for frozen cells is between 40-2 x 10⁵ ... [摘要] TaqMan基因表达细胞至Ct TM TM试剂盒能够直接从细胞裂解物进行逆转录，而无需分离RNA。裂解反应的推荐输入范围在10-10-5个细胞之间，尽管上限可能根据细胞类型而有所不同。该协议部分适用于制造商的协议（ TaqMan Gene Expression Cells-to用于基因表达以便与冷冻细胞团粒一起使用以允许实验室之间的样品转移并涉及在裂解之前通过实验证实的稀释。在这个限制范围内，冷冻细胞的样品细胞数量的推荐范围在40-2×10-5个细胞之间。该试剂盒还设计用于'自制'qPCR主混合物，因为试剂盒中包含的TaqMan Genotyping Master Mix体积可能不足以用于大量样品和/或分析的分析（Smythe和Copren，2008）。细胞计数由荧光激活细胞分选（FACS）提供，并且是该方案的独特方面。【背景】此协议是对TaqMan Gene的修改和实验验证， Expression Cells-to -CT 试剂盒用户指南，出版号为4385117版本E（2012）（Thermo Fisher Scientific / Life ...

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