Preparation of Genomic DNA from Bacteria

• 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)

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 Gel^TM 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.