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RNA-free Plasmid DNA Isolation Protocol

  • Add 1.5 ml TE, (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) containing 50 g/ml RNase A and 2.5 g/ml RNase T1 to the pellet and mix gently to dissolve pellet.
  • Centrifuge for 12 minutes at 10,000 x g to consolidate sample, then transfer sample to a pre-spun (1500 x g for 12 minutes) PLG 15 ml Light tube.
    Note: 510 l of this suspension can be analyzed by 1% agarose gel electrophoresis to confirm that plasmid DNA is present.
  • Incubate for 15 minutes at 37C in a water bath.
  • Extract sample once with 2.0 ml PCI (Phenol-Chloroform-lsoamyl Alcohol 25:24:1). Thoroughly mix the aqueous and organic phases by repeated inversion. Do not vortex. Centrifuge at 1500 x g for 5 minutes to separate the phases.
  • Add 2.0 ml PCI to the aqueous sample in the same PLG 15 ml tube and extract as in step 14.
  • Add 2.0 ml Chloroform-Isoamyl Alcohol (24:1) to the aqueous sample in the same PLG 15 ml tube and extract as in step 14.
  • Carefully transfer resultant aqueous phase (1.41.5 ml) to a suitable fresh tube and process or precipitate the sample as per your protocols and procedures.

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