An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments.

Abstract

The high-throughput, cost-efficient transformation systems determine the success of gene cloning and functional analysis. Among various factors that affect this transformation systems, the competence ability of target cells is one of the most important factors. We found antimicrobial peptides LFcin-B can increase the permeability of the cell membrane, and their lethal antibacterial properties can be inhibited by moderately high concentrations of Ca2+ and Mn2+. In this study, we established a convenient and rapid method (CRM) by adding small concentrations of (0.35mg/L) and moderately high concentrations of MnCl2 (50mM) and CaCl2 (30mM) in transformation buffer. The transformation efficiency of E.coli cells (DH5α, JM109 and TOP10) prepared by CRM were comparable with electroporation for plasmid transformation (3.1 ± 0.3 × 109 cfu/µg). Unlike competent cells prepared using other chemical methods, those obtained using CRM method are extremely competent for receiving larger size DNA fragments (> 5000bp) into plasmid vectors. The competent E.coli cells prepared by CRM method are particularly useful for most high-efficiency transformation experiments under normal laboratory conditions.