Bacterial Transformation
1. Remove one vial containing competent cells (50 µL) from the −80 °C freezer and thaw on ice for 20 minutes.
2. Add plasmid DNA (50–200 ng in a final volume of 1 µL) to the competent cells and mix gently by vortexing. Incubate the mixture on ice for 30 minutes.
3. Heat-shock the cells at 42 °C for 90 seconds.
4. Immediately place the cells on ice and incubate for 5 minutes.
5. Add 350 µL of SOC medium and incubate at 37 °C for 1 hour with shaking at 200 rpm. Keep the tube inclined at approximately 30° to prevent cell sedimentation at the bottom of the tube.
6. Plate 50 µL of the culture onto selective agar plates appropriate for the plasmid used (e.g., LB agar supplemented with the corresponding antibiotic). Spread the cells using sterile beads or a spreader and incubate at 37 °C for 24 hours.
Note: For plasmids with low transformation efficiency, the entire recovery volume may be centrifuged at 1000 rpm for 10 minutes. Discard the supernatant, leaving approximately 100 µL of remaining volume. Resuspend the cells and plate the entire suspension onto a selective agar plate.
7. Count the colony-forming units (CFUs).
Transformation efficiency, expressed as the number of transformed cells per µg of plasmid DNA, can be calculated using the following equation:
Transformation efficiency, expressed as the number of transformed cells per µg of plasmid DNA, can be calculated using the following equation: