This article describes a method for transforming E. coli using electroporation to introduce plasmid DNA. The process involves preparing electrocompetent cells, applying an electric pulse, and selecting transformants on antibiotic-containing agar plates.
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 pre-culture one to 500 in 25 milliliters of fresh LB media and incubate the culture at 37 degrees Celsius until it reaches log phase 0.6 OD. Centrifuge the cell suspension at 5,000 RPM for 20 minutes.
After centrifugation, resuspend the pellet in 25 milliliters of 10% volume per volume, ice-cold, sterile glycerol water, and repeat this step four times, having the resuspension volume each time until 1.5 milliliters is reached.
Next, resuspend the pellet in 10% glycerol and divide the cell suspension in 50 microliter aliquots. Store the aliquots at negative 80 degrees Celsius for up to six months. To continue the experiment, dissolve the desired plasmid in sterile water supplemented with 0.5 millimolar EDTA, and thaw an aliquot of electrocompetent cells on ice.
Add 2.5 to 5 nanograms of vector and mix the thawed cells. Dispense the cells into a 0.1-centimeter cuvette. Apply a 1.8 kilovolt pulse to the cells, and immediately transfer the electroporated cells into one milliliter of SOC media.
Incubate the cells while shaking for one hour, transfer 100 microliter aliquots to petri dishes containing LB agar and antibiotic, and incubate the cells overnight at 37 degrees Celsius. Purify the transformants by streaking single colonies on Petri dishes.
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