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
- 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
- Use a sterile toothpick to streak frozen cells onto a 2X YT + Amp agar plate. Incubate at 37° overnight.
- Autoclave 50 ml 2X YT in a 250 ml flask. Make at least 2 flasks.
- 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.
- Transfer each culture to an Oakridge tube and spin at 8K rpm for 10 minutes in the Sorval with SS-34 rotor.
- 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
- Let sit 5 min, at room temperature.
- 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
- 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
- Spin 10K rpm, 15 min, 4° in the SS34 rotor.
- Transfer supernatant to sterile 125ml flask; add at least 2 volumes of cold 95% EtOH; let stand at least 10 min at room temperature.
- 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.
- 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)
- Resuspend the dry pellet in TE
TLA100.3 VTI65 2.5 ml 4 ml
- Add CsCl to resuspended plasmid, allow it to dissolve
TLA100.3 VTI65 2.5 g 4.21 g
- Add 10mg/ml EtBr to each Quick Seal tube
1/2″ x 1 1/4″ 1/2″ x 2″ 200 µl 270 µl
- Transfer CsCl solution to tube with a Pasteur pipet; balance (paired tubes must not differ by more than 0.1 g); seal.
- Spin at 22°, for at least 8 hours
TLA100.3 VTI65 75k rpm 45k rpm
- Pull of bottom band (closed circle DNA) with a 1ml syringe and an 18 gauge needle.
- Extract at least 4 times with equal volumes of NaCl saturated isopropanol (until the isopropanol is clear).
- 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.
- Vortex, put at 4° for at least 1 hr.
- Spin 15 min at 4°, wash with 70% EtOH, dry in the Speedvac.
- Resuspend in 0.4 ml H20, combining samples that had been divided.
- Add 0.1 ml 7.5M NH4OAc and 1 ml 95% EtOH.
- Leave at -20° overnight.
- Spin 15 min at 4°.
- Wash with 70% EtOH, dry in the Speedvac.
- Resuspend in TE.
- Check OD260
OD260 * 50 * dilution * 10-3 = µg/µl
This page is, as always, Copyright (c) 1998 by Craig D. Amundsen. All rights reserved.