One step 'miniprep' method for the isolation of plasmid DNA protocol

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Solutions/reagents:

Equipment:

  • Centrifuge
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Measure out 0.5 ml of overnight E.coli culture into sterile 1.5-ml microcentrifuge tube (1).
    We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.
  2. Measure out 0.5 ml of phenol : chloroform : isoamyl alcohol(25:24:1) into sterile 1.5-ml microcentrifuge tube (1).
  3. Vortex the mixture for 1 min .
    Vortex at maximum speed.
    Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.
  4. Centrifuge at a speed of 12000 Xg for 5 mins at room temperature.
  5. Meanwhile:
    Set aside a fresh sterile 1.5-ml microcentrifuge tube (2). Call it Tube I.
    Measure out 0.5 ml of isopropanol into Tube I.
  6. Add 0.45 ml of top aqueous phase obtained after centrifugation.
    Vortex the mixture for a few secs.
    Centrifuge at a speed of 12000 Xg for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
    Addition of salt and cooling is unnecessary.
  7. Add 0.5 ml of 70% ethanol.
    Add ethanol carefully to the side of the tube.
    Discard solution.
  8. Repeat Step 7.
    Add 100 µl/ml RNAse to solution.
    Resuspend pellet by vortexing/by shaking vigorously.
    About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 11 mins

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