Small Scale Plasmid Isolation (Miniprep) protocol

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Revision as of 03:45, 23 October 2009 by Vaishnavi Ananth (Talk | contribs)
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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
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Cell Lysis

    1. Measure out 1.5 ml of overnight culture into sterile 1.5-ml microcentrifuge tube (1).
      Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
    2. Add 1.5 ml of overnight culture.
      Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
    3. Add 100 µl of ice-cold Solution I.
      Resuspend ice-cold Solution I by vortexing/by shaking vigorously.
    4. Add 200 µl of Solution II.
      Close the tube tightly and gently mix the contents by inverting the tube.
      Store the tube on ice for 5 mins.
    5. Add 150 µl of Solution III.
      Vortex the mixture for a few secs.
      Store the tube on ice for 10 mins.
    6. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2).
      Discard bottom layer.
  2. Phenol/Chloroform Cleanup

    1. Add 0.5 volume phenol to sterile 1.5-ml microcentrifuge tube (2).
      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 1.5-ml microcentrifuge tube (3).
      Discard bottom layer.
  3. RNase (Optional)

    1. Measure out RNase to a final concentration of 3µg/ml into sterile 1.5-ml microcentrifuge tube (3).
    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 1.5-ml microcentrifuge tube (4).
      Discard bottom layer.

  4. Alcohol Precipitation / Purification

    1. Add 1 volume ice-cold isopropanol to sterile 1.5-ml microcentrifuge tube (4).
    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 200 µl 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 30 - 50 µl of sterile distilled water.
      Resuspend sterile distilled water by vortexing/by shaking vigorously.
    8. Store at -20°C.

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