Kafatos:Minipreps protocol

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Revision as of 01:47, 20 November 2009 by Vaishnavi Ananth (Talk | contribs)
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Solutions/reagents:

  • 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
  • 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 pellet 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 pellet 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.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 7 mins

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