Preparation and Transformation of Bacteria Using High-Voltage Electroporation

Start with a tube containing bacteria inoculated into a nutrient medium.

Incubate with shaking until the cells reach the actively dividing phase.

Centrifuge to collect the cells and discard the supernatant.

Repeatedly wash the cells with ice-cold glycerol to remove salts and resuspend in glycerol to maintain cell viability.

Prepare a plasmid containing an antibiotic resistance gene in sterile water with EDTA to prevent DNA degradation.

Mix the plasmid solution with the bacterial cells.

Apply a short, high-voltage pulse to temporarily create pores in the bacterial membrane.

This allows the plasmid DNA to enter the cytoplasm.

Immediately transfer the cells to a recovery medium and incubate with shaking.

The medium's components support membrane repair and plasmid-encoded antibiotic resistance gene expression.

Spread the cells on nutrient agar containing the antibiotic and incubate until colonies form.

Pick a single colony and streak it onto a fresh plate to purify the transformants.

To begin the experiment, prepare electrocompetent cells of the top 10 E. coli strain.

Transfer a single colony to one milliliter of LB media. Incubate the tube at 37 degrees Celsius while shaking at 180 RPM. Dilute the