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DNA Isolations and Purification

Small Scale DNA Isolation

  1. Grind freeze dried tissue in 2mL tube.
  2. Add 700uL CTAB to tissue, vortex.
  3. Incubate at 65°C for 60-90 minutes, invert every 15 min.
  4. In fume hood, add 400uL Chloroform:IA (24:1), vortex.
  5. Spin 5 minutes.
  6. Transfer top phase to a new 1.6mL tube.
  7. Add 400uL Chloroform:IA, vortex.
  8. Spin 5 minutes.
  9. Transfer top phase to new 1.6mL tube.
  10. Add 300uL Isopropanol, mix by inversion.
  11. Spin 5 minutes, pour off top phase.
  12. Add 500uL 70% EtOH, vortex.
  13. Spin 5 minutes.
  14. Pour off top phase, air dry.
  15. Resuspend in 250uL water or low Tris (pH8)

 

Large Scale DNA Isolation

  1. Perform in a fume hood with protective goggles.
  2. Add 300-400 mg dry tissue to a 15mL Falcon Tube (screw cap).
  3. Add 7 mL CTAB stock solution (with freshly  added β-mercaptoethanol). Vortex to mix well.
  4. Incubate 90 min. at 65°C. Invert every 15 minutes.
  5. Remove tubes from the bath and cool 10 min.
  6. Add 8mL 24:1 Chloroform: IsoAmyl alcohol.
  7. Invert for 10 min. on the rocker.
  8. Centrifuge for 10 min. at 3,700 rpm.
  9. Pour off the supernatant into a new snap-top tube.
  10. Add 5uL of 20 mg/mL Rnase A. Incubate 30 min. at 37°C.
  11. Add 4 ml C/I and shake 10 min.
  12. Centrifuge for 10 min. at 3,700 rpm.
  13. Remove the supernatant with transfer pipette into a new snap-top tube.
  14. Add 5 ml isopropanol to precipitate DNA.
  15.  Remove DNA with glass hook or spin down to pellet the precipitated DNA
  16. Re-suspend DNA in 300-500ul nuclease-free water.
  17. Transfer to a 1.5mL tube.
  18. Quantify DNA using a nanodrop.

 

DNA Cleanup

  1.  Add equal volume of phenol:chloroform to DNA sample and mix well/vortex gently.
  2. Spin at 8,000rpm for 3 minutes.
  3. Transfer the aqueous layer to a new 1.5mL tube and add equal volume chloroform.
  4. Spin at 8,000rpm for 3 minutes.
  5. Transfer the aqueous layer to a new 1.5mL tube.
  6. Add 0.1 volume cold 3M Na-Acetate (pH 5.5) and 2 volumes cold 100% ethanol.  Incubate for 30 minutes to 1 hour at -20°C.
  7. Spin at 10,000rpm for 10 minutes, 4°C.
  8. Remove supernatant and wash with 400uL cold 70% ethanol.
  9. Spin at 10,000rpm for 5 minutes and remove ethanol.
  10. Air dry for 5-10 minutes.
  11. Resuspend DNA in 200uL nuclease-free water.
  12. Quantify DNA using a nanodrop.