使用甘油/甘露醇密度梯度离心法制备高效电转化大肠杆菌感受态细胞。

Preparation of highly efficient electrocompetent Escherichia coli using glycerol/mannitol density step centrifugation.

机构信息

Department of Medical Biochemistry at Radiumhospitalet, Oslo University Hospital, Montebello, N-0310 Oslo, Norway.

出版信息

Anal Biochem. 2011 Jun 15;413(2):206-7. doi: 10.1016/j.ab.2011.02.036. Epub 2011 Apr 6.

DOI:10.1016/j.ab.2011.02.036

Abstract

Traditional protocols for preparing Escherichia coli for electroporation are laborious and often deliver highly variable transformation efficiencies. Many laboratories resort to purchasing expensive commercially prepared cells. This article describes a simple method for producing electrocompetent E. coli by centrifuging bacteria through a glycerol/mannitol density cushion. The method is rapid and replaces tedious multistep procedures with two 15-min centrifugations. Standard cloning strains consistently produce more than 8×10(9)transformants/μg pUC18, whereas the strains TG1 and LE392 display efficiencies of more than 3×10(10)/μg DNA.

摘要

传统的大肠杆菌电穿孔准备方案繁琐，且转化效率通常变化较大。许多实验室转而购买昂贵的商品化制备细胞。本文描述了一种通过甘油/甘露醇密度梯度离心制备电感受态大肠杆菌的简单方法。该方法快速，用两步 15 分钟的离心取代了繁琐的多步操作。标准克隆菌株的转化效率均超过 8×10(9)转化子/μg pUC18，而 TG1 和 LE392 株的转化效率超过 3×10(10)/μg DNA。