Plasmid MAXI prep

  • Grow E. coli in 100 ml LB + antibiotic (e.g. 100ug/ml ampicillin final).
  • Spin 5000 RPM  or more for 10min and just keep pellet.
  • Add 5 ml of solution I ( glucose 0.450 g , 1.25 ml of Tris-pH8 1M,  1 ml of EDTA-pH8 0.5 M, up to 50 ml water) and vortex to have no clumps.  (50 mM glucose, 25 mM Tris-pH8, 10 mM EDTA-pH8).
  • Add 5 ml of fresh solution II (1 ml of NaOH 10N, 2.5 ml of SDS 20%, up to 50 ml water) and invert gently several times  (never vortex and not more than 5 min). (0.2 M NaOH, 1% SDS).
  • Add 5 ml of ice cold solution III (14.7 g KOAc, 5.75 ml glacial acetic acid, up to 50 ml water) and invert gently several times to mix and put on ice for 10 min.
  • Spin 5000 RPM or more for  15 min. For Vial, 12000 RPM or more.
  • Put a piece of cheesecloth (gauze) at the end of a barrel of a 20 ml syringe and pure the supernatant inside it and install its plunger and push it and collect the pass-through in a tube.
  • Add 0.6 volumes ISO-propanol, invert to mix well and leave 10 min at RT.
  • Spin 4000 RPM or more for 15 min.
  • Re-suspend pellet in 700 ul TE-pH8 and transfer it to a 1.5 vial and add 700 ul of ice-cold 5M LiCl and mix well by inverting and keep on ice for 10 min.
  • Spin 10000 RPM 10 min and keep supernatant.
  • Add 0.6 volumes ISO-propanol to supernatant, invert to mix well and leave 10 min at RT.
  • Spin 10000 10 min and keep pellet.
  • Add 250 ul of TE-pH8 and add 5 ul RNase cocktail and keep at 37 for 30 min.
  • Add 240 ul of Proteinase K buffer and 10 ul/ml of Proteinase K and keep at 37 min.
  • Add 300 ul of phenol-pH8 and vortex horizontally for 15s and keep for 5 min at RT. Then add 100 ul chloroform and vortex horizontally for 15s and keep for 5 min at RT.. Spin 10000 RPM for 10 min and keep supernatant.
  • Add the same volume of ISO-propanol, vortex and keep 10 min RT, then spin 10000 RPM 10 min.
  • Add 1 ml of 70 % ethanol, vortex 5-10s and spin 8000 RPM for 1 min.
  • Dissolve the pellet in 1 ml of sterile water and add 0.5 ml of solution (40% PEG 8000/30 Mm MgCl2). Let at RT >10 min and then spin max speed for 20 min at RT.
  • Re-suspend in 0.5 ml 70% Ethanol and spin again for 5 min max speed. Drain, invert and repeat.
  • Add 200 ul DNase free water or TE-pH8 directly on pellet and after dissolving, transfer it to new vial.
Advertisements