A HIGHLY EFFICIENT AND HIGHLY RELIABLE PROTOCOL FOR TRANSFORMATION OF ESCHERICHIA COLI BY ELECTROPORATION
Abstract
ABSTRACT
Electroporation is widely adopted for highly efficient transformation of Escherichia coli of different purposes, but its efficiency is highly varied in the range of several orders of magnitude. Here we describe a modified and highly reliable electroporation protocol which combines low temperature growth in the presence of sucrose with a postelectroporation heat pulse, which not only gives rise to an over 250-fold enhancement in transformation compared with conventional protocols, but also alleviates the need for a strict low temperature control at all times of an electroporation-competent cell preparation experiment and highly pure MilliQ grade water which is not readily available in many laboratories. Transformations for a 6-kb E. coli and yeast shuttle vector were always higher than 107 transformants/µg, and more than 108 transformants/µg were sometimes recorded in routine experiments.
PRACTICAL APPLICATIONS
Transformation efficiency reached up to 8 × 109 transformants/µg DNA with ElectroMAXTM DH10BTMEscherichia coli cells and a 6-kb pYES2/CT plasmid using our procedure, when combined with reduction of ampicillin concentration from 100 µg/mL to 20 µg/mL in the selection plates. It is highly efficient and highly reliable on DNA library construction of all kinds, especially from minute cell and tissue samples, under routine laboratory conditions. It will find widespread application in molecular manipulations in Escherichia coli, the workhorse of molecular biology.
