Preparation and Transformation of Bacteria Using High-Voltage Electroporation

Abstract

Source: Stefan, A., et al., The Multifaceted Benefits of Protein Co-expression in Escherichia coli. J. Vis. Exp. (2015)

This video demonstrates the procedure for transforming Escherichia coli with a plasmid carrying an antibiotic resistance gene, including cell preparation, electroporation, recovery, and selection of transformants.

Protocol

1. Isolation of E. coli Co-transformants

1. Prepare electro-competent cells of the appropriate E. coli strain to be transformed. Transfer to 1 ml of Luria-Bertani (LB) medium (Tryptone, Yeast Extract, NaCl at 10, 5, and 10 g/L, respectively) a single colony of the strain of choice, and incubate at 37 °C under shaking conditions of 180 rpm. Dilute the pre-culture 1:500 in 25 ml of fresh LB medium and incubate the culture at 37 °C.
2. At log-phase (0.6 O.D. or optical density), centrifuge the cell suspension at 5,000 x g for 20 min, and resuspend the pellet in 10%, v/v ice-cold sterile glycerol-water in half of the original culture volume. Repeat this st....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Sigma-Aldrich	A1296
Ampicillin	Sigma-Aldrich	A9518
EDTA	Sigma-Aldrich	EDS
Glycerol	Sigma-Aldrich	G5516
KCl	Sigma-Aldrich	P9541
MgCl₂	Sigma-Aldrich	M2670
NaCl	Sigma-Aldrich	31434
Tryptone	Sigma-Aldrich	95039
Yeast extract	Fluka	70161
Cuvettes 0.1 cm	BioRad	1652089	For electroporation
Centrifuge 5415R	Eppendorf
Centrifuge Allegra 21R	Beckman
Gene Pulser II electroporation	BioRad