- TE buffer
- 10% (w/v) sodium dodecyl sulfate (SDS)
- 20 mg/mL proteinase K
- Phenol/chloroform (50:50)
- 70% ethanol
- 3M sodium| acetate pH 5.2 phase Lock gel (5 prime, 3 prime)
- 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.
- 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
- 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.
- 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.
- Add 1/10 volume of sodium acetate mix.
- Add 0.6 volume of isopropanol and mix gently until the DNA gets precipitates.
- Spool DNA onto a glass rod (or Pasteur pipette with a heat-sealed end).
- Wash DNA by dipping end of rod into 1 mL of 70% ethanol for 30
- Resuspend DNA in a 100–200 µL TE buffer. Complete resuspension may
take several days.
- Store DNA at 4°C short term, –20°C or –80°C long term.
- After DNA has dissolved, determining the concentration by measuring the
absorbance at 260 nm.