Large Scale Amplification of Plasmid DNA

Large Scale Amplification of Plasmid DNA

  1. Streak colony of E. coli with the plasmid on CA containing the appropriate antibiotic (usually Tet, Crb, or Amp). Incubate at 37 C.
  2. The next day, inoculate a LB flask (with the appropriate antibiotic) and shake at 350 rpm. Put the antibiotic in 6 or 12 small LB flasks and 6 or 12 Fernback flasks containing 500 ml LB (put in incubator to get warm).
    Crb (at 100 mg/ml): 0.5 ml/L, 0.25 ml/0.5 L, 12.5 µl/25 ml
    Tet (at 12.5 mg/ml) : 1.2 ml/L, 0.6 ml/0.5 L, 30 µl/25 ml
  3. Early on day three, put about 0.5 ml (use sterile DPTP) of the overnight culture into each pre-warm small LB flasks (and a few drops in a glycerol tube if a stock is needed). Let these shake (350 rpm) until fairly cloudy (3 hr usually).
  4. Pour each of the small flasks into each of the Fernback flask and shake for 2.5 hr. Then add 2.5 ml of 34 mg/ml chloramphenicol stock solution (in ethanol) and shake overnight.
    Note: Use gloves when working with powdered or concentrated solutions of chloramphenicol: it is suspected carcinogen.
  5. The next morning spin the cells down in a GSA rotor 7,000 rpm, 5 min.
  6. Wash and consolidate the cells by scraping and resuspending them in some saline and give them another spin in a single bottle.
  7. For 3 L of cells (up to 14 g), suspend well in 136 ml (final volume) cold (i.e. on ice) 50:50 TE with 15 % sucrose. Dissolve about 50 mg lysozyme in 4 ml of buffer and add to the cells, mixing by inversion. Put 17 ml in each of 8 cold (i.e. on ice) polycarbonate 70 Ti centrifuge tubes.
  8. Violently squirt in 3.5 ml of 10 % SDS and immediately violently squirt 4.5 ml saturated NaCl, cap, the tube, mix by violent inversion, and put back on ice: do all at each tube one at a time.
  9. Let the tube sit on ice (or in the cold 70 Ti rotor) for at least one hr. Spin 30 min at 30,000 rpm at 4 C.
  10. Pool the supernatant into two 250 ml centrifuge bottles (look for good ones and use the tops with many ridges) and phenol:CHCl3 extract it giving it NH4Ac/EtOH precipitation at the end.
  11. Give the sample a CsCl/EtBr density gradient spin.