Small Scale Plasmid Isolation (Maxiprep) protocol

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Solutions/reagents:

  • overnight culture
  • ice-cold Solution I
  • Solution II
  • Solution III
  • phenol
  • chloroform
  • RNase to a final concentration of 3µg/ml
  • ice-cold isopropanol
  • 70% ethanol
  • sterile distilled water

Equipment:

  • Centrifuge
  • Incubator
  • 50-ml centrifuge tubes
  • Sterile 15-ml centrifuge tubes

Steps:

  1. Cell Lysis

    1. Measure out 50 ml of overnight culture into 50-ml centrifuge tube (1).
      Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.

    2. Add 1 ml of ice-cold Solution I.
      Resuspend pellet by vortexing/by shaking vigorously.

    3. Add 2 ml of Solution II.
      Close the tube tightly and gently mix the contents by inverting the tube.
      Store the tube on ice for 5 mins.

    4. Add 1.5 ml of Solution III.
      Vortex the mixture for a few secs.
      Store the tube on ice for 10 mins.

    5. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (1).
      Discard bottom layer.

  2. Phenol/Chloroform Cleanup

    1. Add 0.5 volume phenol to sterile 15-ml centrifuge tube (1).
      Add 0.5 volume chloroform.

    2. Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (2).
      Discard bottom layer.

    3. RNase (Optional)

    1. Measure out RNase to a final concentration of 3µg/ml into sterile 15-ml centrifuge tube (2).

    2. Incubate at 37°C for 30 - 45 mins.
      Use a water bath.

    3. Add 0.5 volume phenol.
      Add 0.5 volume chloroform.
      Vortex the mixture for a few secs.
      Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 15-ml centrifuge tube (3).
      Discard bottom layer.

  3. Alcohol Precipitation / Purification

    1. Add 1 volume ice-cold isopropanol to sterile 15-ml centrifuge tube (3).

    2. Close the tube tightly and gently mix the contents by inverting the tube.
      Store the tube on ice for 10 mins.

    3. Centrifuge at maximum speed for 15 mins at room temperature, gently aspirate out the supernatant and discard it.

    4. Add 2 ml of 70% ethanol.
      Mix solution by pipetting up and down several times.

    5. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.

    6. Dry the pellet in air for at most 30 mins.

    7. Add 200 - 300 µl of sterile distilled water.
      Resuspend pellet by vortexing/by shaking vigorously.

    8. Store at -20°C.

    TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 2 hrs, 20 mins

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