ORS571 DNA EXTRACTION PROCEDURE - MODIFIED FOR TL-100

ORS571 DNA EXTRACTION PROCEDURE - MODIFIED FOR TL-100

  1. Grow cells overnight in 40.0 ml or more GYPC broth medium.
  2. Pellet cells by centrifugation, and resuspend in 1 ml 50 mM Tris (pH 8.0), 50 mM EDTA.
  3. Freeze cell suspension at -20C (optional).
  4. Add 0.1 ml 250 mM Tris (pH 8.0), 10 mg/ml lysozyme to frozen suspension (if frozen), and let thaw at room temperature. When thawed, place on ice for 45 min.
  5. Add 0.2 ml 50 mM Tris (pH 7.5), 0.4 M EDTA, 1 mg/ml proteinase K. Then add 0.2 ml 10% sarcosyl and mix gently. Place in 50C water bath for 10 min. Check for cell lysis. Add RNase to 200 g/ml and incubate for 10 more minutes.
  6. Extract with 2 ml Tris-equilibrated phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuge at 10,000X rpm for 20 min in JA-18 rotor at 4C. Transfer top layer to new tube (avoid interface). Re-do this step if necessary.
  7. Adjust aqueous phase to 1.7 ml with TE buffer. To this add 2.0 g CsCl and 80 l ethidium bromide (10 mg/ml); the total volume should be approximately 2.2 ml. If necessary, CsCl solution (1 mg/ml) should be added to completely fill each tube. Note: Empty 2.2 ml quick seal tubes weigh about 0.5 grams. Filled tubes with 1.6 g/ml density should be about 4.0 grams.
  8. Place in 2.2 ml Quick-seal tubes, seal, and centrifuge in TLA-100.2 rotor at 100,000 rpm at 20C for at least 4 hrs, depending on the resolution required). Quick-seal tubes in the TLA-100.2 rotor require tube caps.
  9. Remove tubes carefully from rotor. Puncture top of tube with a needle. Visualize DNA band by longwave uv light and pull off DNA bands with a 1cc syringe and 18G needle.
  10. Extract out ethidium bromide from DNA sample with equal volume of 5 M NaCl, TE saturated isopropanol. Remove top isopropanol phase and discard. Repeat extraction until solution is clear in color. Add 2 volumes water and 6 volumes 95% ethanol to extracted aqueous phase, then place at 20 C for 3 hours or overnight.
  11. Centrifuge at 10000 rpm in a JA18 rotor for 15 min at 4 C, decant supernatant, wash with cold 70% ethanol, recentrifuge, decant supernatant, vacuum dry pellet, and resuspend in 100 ul TE.