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Roche Applied Science: High Pure PCR Purification Kit

Protocol obtained from: High Pure PCR Purification Kit

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The following protocol is for the purification procedure for DNA from a 100 mg agarose gel slice

  1. Isolate DNA band of interest electrophoretically as follows.
    • Load PCR reaction mixture on a 0.8 - 2% agarose gel.
    • Use 1 × TAE or 1 × TBE as running buffer.
    • Electrophorese until DNA band of interest is isolated from adjacent contaminating fragments
  2. Identify bands by staining gel with ethidium bromide or SYBR Green I or Nucleic Acid Gel Stain.
    • VAdv.jpgWear gloves, ethidium bromide is a known potent carcinogen.
  3. Cut desired DNA band from gel using an ethanol-cleaned scalpel or razor blade.
    • VAdv.jpgMinimize gel volume by visualizing DNA and cutting the smallest possible gel slice on a UV light box.
  4. Place excised agarose gel slice in a sterile 1.5 ml microcentrifuge tube.
    • Determine gel mass by first pre-weighting the tube, and then reweighting the tube with the excised gel slice.
  5. Add 300 μl Binding Buffer for every 100 mg agarose gel slice to the microcentrifuge tube.
  6. Dissolve agarose gel slice in order to release the DNA:
    • Vortex the microcentrifuge tube 15 - 30 s to resuspend the gel slice in the Binding Buffer.
    • Incubate the suspension for 10 min at 56°C.
    • Vortex the tube briefly every 2 - 3 min during incubation.
  7. After the agarose gel slice is completely dissolved:
    • Add 150 μl isopropanol for every 100 mg agarose gel slice to the tube.
    • Vortex thoroughly.
  8. Insert one High Pure Filter Tube into one Collection Tube.
    • Pipette the entire contents of the microcentrifuge tube into the upper reservoir of the Filter Tube.
    • VAdv.jpgDo not exceed 700 μl total volume. If mixture is > 700 μl, split the volume and use two separate Filter Tubes for each portion.
  9. Centrifuge 30 - 60 s at maximum speed in a standard table top centrifuge at +15 to +25°C.
    • Discard the flowthrough solution.
    • Reconnect Filter Tube with the same Collection Tube.
  10. Add 500 μl Wash Buffer to the upper reservoir.
    • Centrifuge 1 min at maximum speed (as above).
  11. Discard the flowthrough solution.
    • Recombine Filter Tube with the same Collection Tube.
    • Add 200 μl Wash Buffer.
    • Centrifuge 1 min at maximum speed.
    • VLupa.jpgThis second 200 μl wash step ensures optimal purity and completeremoval of Wash Buffer from the glass fibers.
  12. Discard the flowthrough solution and Collection Tube.
    • Recombine Filter Tube with a clean 1.5 ml microcentrifuge tube.
  13. Add 50 - 100 �l Elution Buffer to the upper reservoir of the Filter Tube.
    • Centrifuge 1 min at maximum speed.
  14. The microcentrifuge tube now contains the purified DNA.
    • VAdv.jpgWhen subsequent OD260 determination is planned, centrifuge the eluate for more than 1 min at maximum speed to remove residual glass fibers from the eluate, because they may disturb absorbance measurement. Use an aliquot of the supernatant to determine concentration.
    • VLupa.jpgEither use the eluted DNA directly or store the eluted DNA at +2 to +8°C or -15 to -25°C for later analysis.