CsCl Plasmid Prep

CsCl Plasmid Prep

For the TLA100.3 (1/2″ x 1 1/4″ Quick Seal tubes)
and VTI65 (1/2″ x 2″ Quick Seal tubes) Rotors
(Maniatis based)

Schedule:

  • Day -1: Grow cells on a selection plate overnight
  • Day 0: Grow single colonies of cells in liquid, under selction overnight
  • Day 1: Prepare ultracentrifuge tubes; spin overnight
  • Day 2: Isolate DNA from CsCl solution; preciptate overnight
  • Day 3: Wash, dry, and resuspend plasmid DNA

Protocol:

  1. Use a sterile toothpick to streak frozen cells onto a 2X YT + Amp agar plate. Incubate at 37° overnight.
  2. Autoclave 50 ml 2X YT in a 250 ml flask. Make at least 2 flasks.
  3. Add 50 to 100 µl of 50 mg/ml Amp to each flask. Pick a single colony of cells into each flask of media; incubate at 37°, with shaking, overnight.
  4. Transfer each culture to an Oakridge tube and spin at 8K rpm for 10 minutes in the Sorval with SS-34 rotor.
  5. Resuspend each pellet of cells in 4ml TEG using a Pasteur pipette
    [Stock] Reagent Amount [Final]
    1 M Tris (pH = 8) 2.0 ml 25 mM
    0.5 M EDTA 0.8 ml 10 mM
    2 M Glucose 0.8 ml 50 mM
    – ddH2O 36.7 ml –
    Total 40 ml
  6. Let sit 5 min, at room temperature.
  7. Add 8ml fresh lysis solution to each tube; invert to mix; let sit 10 min, on ice.
    [Stock] Reagent Amount [Final]
    10 N NaOH 1.0 ml 0.2N
    20 % SDS 2.5 ml 1 %
    – H2O 46.5 ml –
    Total: 50 ml
  8. Add 6ml ice-cold 3M K 5M Acetate; invert; vortex; let sit 10 min, on ice.
    Reagent Amount
    KAc 29.44 g
    Glacial Acetic Acid 11.5 ml
    H2O to 100 ml
  9. Spin 10K rpm, 15 min, 4° in the SS34 rotor.
  10. Transfer supernatant to sterile 125ml flask; add at least 2 volumes of cold 95% EtOH; let stand at least 10 min at room temperature.
  11. Spin half of the solution in a sterile 30ml Corex tube, 8K, 10 min in the SS34 rotor; pour off the supernatant; repeat with the remaining half.
  12. Dry the pellet in the Corex tube in the Speedvac (cover with parafilm; poke holes in the film; lay tube in Speedvac under the rotor)
  13. Resuspend the dry pellet in TE
    TLA100.3 VTI65
    2.5 ml 4 ml
  14. Add CsCl to resuspended plasmid, allow it to dissolve
    TLA100.3 VTI65
    2.5 g 4.21 g
  15. Add 10mg/ml EtBr to each Quick Seal tube
    1/2″ x 1 1/4″ 1/2″ x 2″
    200 µl 270 µl
  16. Transfer CsCl solution to tube with a Pasteur pipet; balance (paired tubes must not differ by more than 0.1 g); seal.
  17. Spin at 22°, for at least 8 hours
    TLA100.3 VTI65
    75k rpm 45k rpm
  18. Pull of bottom band (closed circle DNA) with a 1ml syringe and an 18 gauge needle.
  19. Extract at least 4 times with equal volumes of NaCl saturated isopropanol (until the isopropanol is clear).
  20. Divide into 200µl or less aliquots in microfuge tubes; add 3 volumes of H2O, 0.06 new volumes of 7.5M NH4OAc, 0.7 new volumes of isopropanol.
  21. Vortex, put at 4° for at least 1 hr.
  22. Spin 15 min at 4°, wash with 70% EtOH, dry in the Speedvac.
  23. Resuspend in 0.4 ml H20, combining samples that had been divided.
  24. Add 0.1 ml 7.5M NH4OAc and 1 ml 95% EtOH.
  25. Leave at -20° overnight.
  26. Spin 15 min at 4°.
  27. Wash with 70% EtOH, dry in the Speedvac.
  28. Resuspend in TE.
  29. Check OD260

OD260 * 50 * dilution * 10-3 = µg/µl

This page is, as always, Copyright (c) 1998 by Craig D. Amundsen. All rights reserved.