Preparation of Genomic DNA from Bacteria

Materials
  • TE buffer
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 20 mg/mL proteinase K
  • Phenol/chloroform (50:50)
  • Isopropanol
  • 70% ethanol
  • 3M sodium| acetate pH 5.2 phase Lock gel (5 prime, 3 prime)
Procedure
  1. Grow E. coli culture overnight in rich broth. Transfer 1.5 mL to a microcentrifuge tube and spin 2 min. Decant the supernatant. Repeat with another 1.5 mL of cells. Drain well onto a Kimwipe.
  2. Resuspend the pellet in 467 µL TE buffer by repeated pipetting. Add 30 mL of 10% SDS and 3 µL of 20 mg/mL proteinase K, mix, and incubate 1 hr at 37°C.
  3. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
    Caution: Phenol causes severe burns. Wear gloves, goggles, and a lab coat, and keep tubes capped tightly.
    Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin 2 min.
  4. Transfer the upper aqueous layer phase to a new tube and add an equal volume of phenol/chloroform. Again mix well and transfer to a New Phase Lock GelTM tube and spin 5 minutes. Transfer the upper aqueous phase to a new tube.
  5. Add 1/10 volume of sodium acetate mix.
  6. Add 0.6 volume of isopropanol and mix gently until the DNA gets precipitates.
  7. Spool DNA onto a glass rod (or Pasteur pipette with a heat-sealed end).
  8. Wash DNA by dipping end of rod into 1 mL of 70% ethanol for 30 seconds.
  9. Resuspend DNA in a 100–200 µL TE buffer. Complete resuspension may take several days.
  10. Store DNA at 4°C short term, –20°C or –80°C long term.
  11. After DNA has dissolved, determining the concentration by measuring the absorbance at 260 nm.

Support our developers

Buy Us A Coffee

More in this section