Intermediate Scale Preparation of plasmid DNA

Intermediate Scale Preparation of plasmid DNA

  1. Start single colonies of plasmid containing E. coli transformants in 45 ml LB (with 50 µg Crb/ml) and shake overnight at 37 C (350 rpm).
  2. Spin the tubes (10 K, 3 min), and decant supernatent.
  3. Add 5 ml ice cold 50 mM glucose 25:10 TE pH 8 with a scoop of lysozyme, voltex well until resuspended, and let sit 5 min at room temperature.
  4. Add 10 ml freshly made 0.2 N NaOH 1% SDS, invert to mix, and set on ice 5 min.
    * To make up 90 ml for each set of 8 preps: 78 ml water in a flask, mix in 3 ml 6 N NaOH, then mix in 9 ml 10 % SDS.
  5. Add 7.5 ml 3 M K 5 M Ac (ice cold), shake up and down to mix, and set on ice 5 min or more.
  6. Spin the tubes (14 K, 5 min) transfer to new tube, fill with phenol/CHCl3, shake well, and spin (10K, 5 min) to separate the two liquid phases.
  7. Transfer the aqueous phase (top) to a new tube with a DPTP and fill with CH3Cl, invert to mix, and spin (10K, 5 min).
  8. Transfer the aqueous phase (top) to a new tube with a DPTP and fill with isopropanol, shake to mix and let it sit (15 min or longer) (The first good stopping point). Rinse out the rotor to remove any phenol seepage.
  9. Spin the tubes (15 min, 20 K), dump supernatent, rinse with 70 % EtOH, invert tubes to dry on rack with paper towel roll (several min).
  10. Resuspend in 2.1 ml TE and 5ʵl 10 mg/ml RNase (Be sure it is completely dissolved).
  11. Add 700 µl 10 M NH4Ac, mix, let sit on ice at least 10 min.
  12. Spin the tubes (14 K, 20 min).
  13. Pour the supernantent to a corex tube (Take a few seconds to let all the liquid drops transfer, and tap the oakridge tube to get the last drops).
  14. Add 7 ml EtOH, mix, spin (10 K, 15 min).
  15. Dump supernatent, rinse with 70 % EtOH, and dry briefly.
  16. Resuspended pellet (DNA) in 315 µl TE.
  17. Transfer the 315 µl to a MFT, then rinse the corex tube with 105 µl 10 M NH4Ac and 1050 µl EtOH and add to the same MFT.
  18. Mix the solution by invertion and tapping the MFT.
  19. Spin the MFT (12 K, 20 min).
  20. Resuspend DNA pellet in 100 µl TE. (If DNA quantity in MFT is estimated, add TE to get 5 µg DNA/µl TE).
  21. Verify and estimate DNA of the plasmids by diluting the samples and electrophoresis (0.7 %, 100 V, 1 hr).
    * If desired to use a 37 C shaker (50X250 ml flasks) and a centrifuge (20K rpm) with 50 ml oak ridge tubes.