Miniprep - GET Buffer protocol

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Solutions/reagents:

Equipment:

  • Centrifuge
  • Sterile 2-ml microcentrifuge tubes

Steps:

  1. Measure out 2 ml of overnight culture into sterile 2-ml microcentrifuge tube (1).
  2. Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
  3. Measure out 100 µl of ice-cold GET buffer into sterile 2-ml microcentrifuge tube (1).
    Resuspend pellet by vortexing/by shaking vigorously.
  4. (Optional)
    Add 10 mg of lysozyme.
    Store at room temperature for 30 mins.
    This step is essential for lysing gram-positive cells.

  5. Measure out 200 µl of 0.2M NaOH into sterile 2-ml microcentrifuge tube (2).
    Add 2 mg of SDS.
    Vortex the mixture for a few secs.
  6. Measure out alkaline SDS solution into sterile 2-ml microcentrifuge tube (1).
    Close the tube tightly and gently mix the contents by inverting the tube.
    DO NOT VORTEX! The solution should become clear.
  7. Add 150 µl of ice-cold potassium acetate solution.
    Close the tube tightly and gently mix the contents by inverting the tube.
    DO NOT VORTEX! A precipitate should form.
  8. Store the tube on ice for 3 - 5 mins.
  9. Centrifuge at maximum speed for 10 mins at room temperature and aspirate out 400 µl of top layer.
    Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (3).
    Discard bottom layer.
    DO NOT PICK UP ANY PRECIPITATE!!!
  10. (Optional)
    Measure out 400 µl of PCA solution into sterile 2-ml microcentrifuge tube (3).
    Close the tube tightly and gently mix the contents by inverting the tube.
    Centrifuge at maximum speed for 3 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (4).
    Discard bottom layer.
    This helps remove any residual proteins.

  11. Measure out 900 µl of 95% / 100% ethanol into sterile 2-ml microcentrifuge tube (4).
    This is to precipitate the plasmid DNA.
  12. Store at -80°C for 30 mins.
    Use the -80°C freezer.
  13. Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
  14. Add 1 ml of 70% EtOH.
    Store at room temperature for 3 mins.
  15. Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
    Make sure the pellet is toward the outside.
  16. Dry the pellet in air for 10 - 15 mins.
  17. Make sure the pellet is completely dry before this step.

    Option 1: Add 20 µl of TE buffer.
    (or)
    Option 2: Add 20 µl of distilled water.

    Resuspend pellet by vortexing/by shaking vigorously.
    The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 50 mins

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