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RNA-free Plasmid DNA Isolation Protocol

The following protocol is for use with 1 liter cultures grown in LB medium for 1214 hours. It is also used for cultures grown for no longer than 1214 hours in 0.5 liter of enriched growth media such as Terrific Broth (TB). The reagents used in the cleared lysate preparation (step 15) should be scaled down proportionately for smaller culture volumes down to 200 ml.

  1. Pellet bacteria from the culture at 10,000 x g for 5 minutes at 4C.
  2. Resuspend bacterial pellet in a total of 30 ml 25 mM Tris-Cl/10 mM EDTA (pH 8.0). Pipet up and down or vortex as necessary to fully resuspend the bacteria.
  3. Add 30 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex.
  4. Add 30 ml ice-cold 7.5 M Ammonium Acetate to the lysate. Mix thoroughly by repeated gentle inversion. Do not vortex.
  5. Centrifuge at 15,500 x g for 30 minutes at 4C.
  6. Recover resultant supernatant to a fresh centrifuge bottle. Do not carry over any whitish-grey pellet material.
  7. Add 54 ml (0.6 x volume) room temperature 100% Isopropanol to the supernatant. Mix thoroughly by repeated inversion. Do not vortex.
  8. Centrifuge at 15,500 x g for 30 minutes at 20C. Discard resultant supernatant.
  9. Add 25 ml 70% Ethanol to the pellet. Mix by repeated inversion and centrifuge at 15,500 x g for 5 minutes to re-pellet DNA.
  10. Discard resultant supernatant, carefully aspirate any excess Ethanol, and dry the pellet for 1530 mi nutes.
  11. 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.
  12. 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.
  13. Incubate for 15 minutes at 37C in a water bath.
  14. 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.
  15. Add 2.0 ml PCI to the aqueous sample in the same PLG 15 ml tube and extract as in step 14.
  16. 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.
  17. 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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