Bacterial Transformations

Remove an aliquot of frozen chemically competent cells from -80°C storage and thaw on ice for 30 minutes. Simultaneously place LB agar plates containing the appropriate antibiotic on the bench to reach room temperature.

Add 10 picograms to 100 nanograms of plasmid DNA to the thawed competent cells and mix gently by flicking the tube. Prepare a control sample by mixing a separate aliquot of competent cells with water.

Place the tube in a floating rack in a 42°C water bath for 45 seconds to increase cell membrane permeability. Immediately transfer the tube to ice for 2 minutes to stop the heat shock reaction.

Add 500 microliters of SOC media containing glucose to the transformed cells and incubate in a shaking incubator at 37°C for 45 minutes. This allows the bacteria to express antibiotic resistance proteins encoded by the plasmid.

Centrifuge the cells for 5 minutes at 4,000 rpm to concentrate them, then remove some supernatant and resuspend. Pipette the concentrated cells onto warmed LB agar plates containing antibiotic and spread using sterile technique.

Place the agar plates in an incubator at 37°C (or 30°C depending on the bacterial strain) for overnight incubation. Inspect plates the next day for visible colony growth; the control plate should show no colonies.

For fast transformations of easy-to-transform plasmids, skip heat shock and outgrowth steps and incubate the DNA-cell mixture on ice for 5 minutes before plating. For difficult-to-transform constructs larger than 10 kilobases, use electrocompetent cells and electroporation instead of chemical heat shock.