Plasmid DNA Extraction:

Materials Needed:

  • Bacterial culture containing plasmids
  • Plasmid DNA extraction PCR kit
  • Centrifuge
  • Microcentrifuge tubes
  • Isopropanol and ethanol
  • DNA storage buffer

Procedure:

  1. Bacterial Culture:
    • Grow a bacterial culture containing plasmids.
  2. Harvest Cells:
    • Centrifuge the culture to collect bacterial cells.
  3. Resuspend Cells:
    • Resuspend the bacterial pellet in a lysis buffer.
  4. Lysis:
    • Add an alkaline lysis solution to lyse the cells and denature proteins.
  5. Neutralization:
    • Neutralize the lysate to stabilize plasmid DNA.
  6. Centrifugation:
    • Centrifuge to separate cell debris from the cleared lysate.
  7. DNA Precipitation:
    • Precipitate DNA by adding isopropanol.
  8. Washing:
    • Wash the DNA pellet with ethanol.
  9. DNA Resuspension:
    • Resuspend the purified plasmid DNA in a storage buffer.

Plasmid Transformation:

Materials Needed:

  • Competent cells (bacterial cells made permeable for DNA uptake)
  • Plasmid DNA
  • Transformation buffer
  • Heat shock apparatus
  • Recovery medium (LB broth or SOC medium)

Procedure:

  1. Thaw Competent Cells:
    • Thaw competent cells on ice.
  2. Add DNA:
    • Add plasmid DNA to competent cells.
  3. Incubation:
    • Incubate the cells and DNA on ice.
  4. Heat Shock:
    • Subject the cells to a brief heat shock.
  5. Cooling:
    • Cool the cells on ice again.
  6. Recovery:
    • Add recovery medium and incubate the cells at a suitable temperature.
  7. Plating:
    • Plate cells on selective agar plates.
  8. Incubation:
    • Incubate the plates overnight for colony growth.

Plasmid Analysis:

Materials Needed:

  • Agarose gel
  • Gel electrophoresis apparatus
  • DNA ladder
  • Ethidium bromide or other DNA stain
  • UV transilluminator

Procedure:

  1. Prepare Agarose Gel:
    • Cast an agarose gel with appropriate concentration.
  2. Load Samples:
    • Mix plasmid DNA samples with loading dye and load onto the gel.
  3. Run Electrophoresis:
    • Run the gel at a suitable voltage to separate DNA fragments.
  4. Staining:
    • Stain the gel with ethidium bromide or another DNA stain.
  5. Visualization:
    • Visualize DNA bands under a UV transilluminator.