Kafatos:Minipreps protocol

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Solutions/reagents:

  • 5 ml sterile LB medium (+ antibiotic)
  • P1 Buffer
  • P2 Buffer
  • ice-cold P3 Buffer
  • absolute EtOH
  • 70% ethanol
  • TE
  • ddH2O
  • single bacterial colony

Equipment:

  • Incubator
  • Centrifuge
  • Electrophoretic unit
  • Flasks of appropriate volumes
  • Eppendorf tubes

Steps:

  1. DAY 1
    Inoculate 5 ml sterile LB medium (+ antibiotic) with single bacterial colony and incubate with shaking for 12 hrs(overnight) at 37°C.
  2. DAY 2

    1. Measure out 1.5 ml of culture into Eppendorf tube (1).
      Centrifuge at a speed of 8000 rpm for 2 mins at room temperature, gently aspirate out the supernatant and discard it.
    2. Add 100 µl of P1 Buffer.
      Resuspend P1 Buffer by vortexing/by shaking vigorously.
      Store at room temperature for 5 mins.
    3. Add 200 µl of P2 Buffer.
      Store at room temperature for at most 5 mins.
    4. Add 150 µl of ice-cold P3 Buffer.
      Vortex the mixture for a few secs.
      Store the tube on ice for 5 - 10 mins.
    5. Centrifuge at maximum speed for 10 mins at 4°C and aspirate out the top layer.
      Transfer top aqueous layer into Eppendorf tube (2).
      Discard bottom layer.
    6. Add 2 volumes absolute EtOH to Eppendorf tube (2).
      Store the tube on ice for 10 - 20 mins.
    7. Centrifuge at maximum speed for 10 mins at 4°C, gently aspirate out the supernatant and discard it.
    8. Add 1 ml of 70% ethanol.
      Mix solution by pipetting up and down several times.
    9. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  3. Option 1: Dry the pellet in air.
    (or)
    Option 2: Dry the pellet in speedvac.

    Do not overdo drying, or the pellet will not properly dissolve again.

  4. Option 1: Add 30 µl of TE.
    (or)
    Option 2: Add 30 µl of ddH2O.

    Resuspend ddH2O by vortexing/by shaking vigorously.
    Perform agarose gel electrophoresis of appropriate quantity of ddH2O mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.

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