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Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip.

机译:在微流控芯片上从电渗透的大肠杆菌细胞中提取核酸和蛋白质。

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摘要

Due to the extensive use of nucleic acid and protein analysis of bacterial samples, there is a need for simple and rapid extraction protocols for both plasmid DNA and RNA molecules as well as reporter proteins like the green fluorescent protein (GFP). In this report, an electropermeability technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low applied voltages. This will allow small biomolecules with diameters less than 30 A to rapidly diffuse from the permeabilized cells to the surrounding solution. By controlling the applied voltage, partial and transient to complete cell opening can be obtained. By using DC voltages below 0.5 V, cell lysis can be avoided and the transiently formed pores can be closed again and the cells survive. This method has been used to extract RNA and GFP molecules under conditions of electropermeability. Plasmid DNA could be recovered when the applied voltage was increased to 2 V, thus causing complete cell lysis.
机译:由于细菌样品的核酸和蛋白质分析得到了广泛的应用,因此需要简单,快速的提取方案,以用于质粒DNA和RNA分子以及报告蛋白,例如绿色荧光蛋白(GFP)。在此报告中,已经开发了一种电渗透技术,该技术基于将大肠杆菌细胞暴露于低电压下以允许提取核酸和蛋白质。所使用的流通式电导率芯片由带有集成金电极的微流体通道组成,可在低施加电压下促进细胞包膜通道的形成。这将使直径小于30 A的小生物分子从透化的细胞迅速扩散到周围的溶液中。通过控制施加的电压,可以获得部分和瞬态以完全打开电池。通过使用低于0.5 V的DC电压,可以避免细胞裂解,并且可以再次封闭瞬时形成的孔,并使细胞存活。该方法已用于在电导率条件下提取RNA和GFP分子。当施加的电压增加到2 V时,质粒DNA可以回收,从而导致细胞完全裂解。

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