Preparation and Transformation of Bacteria Using High-Voltage Electroporation

Samenvatting

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 step 4 times, halving the resuspension volume. Finally, resuspend the pellet in glycerol/water and divide the cell suspension into 50 μl aliquots. Store the aliquots at -80 °C up to 6 months.
3. Dissolve the desired plasmid in sterile water supplemented with 0.5 mM EDTA (ethylenediaminetetraacetic acid). Thaw an aliquot of electro-competent cells on ice and mix with an appropriate amount (2.5-5 ng) of vector. Dispense the mixture into a 0.1 cm cuvette suitable for electroporation and apply 1.8 kV.
4. Immediately transfer the electroporated cells into 1 ml of SOC (super optimal broth with catabolite repression) medium (LB medium supplemented with 0.2% w/v glucose, 10 mM MgCl₂, 2.5 mM KCl), incubate for 1 hr under shaking, and finally transfer 100 μl aliquots to Petri dishes containing LB agar with the appropriate antibiotic. Incubate O/N at 37 °C.
5. Purify the transformants by streaking single colonies on Petri dishes.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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