LARGE SCALE PLASMID PREP CsCl METHOD

Triton lysis / CsCl method

Grow 500ml cultures with antibiotic selection shaking at 37oC.
When OD600 = 0.8 you may add chloroamphenicol to 125ug/ml.
Let it go overnight.

Spin down the cultures in sterile bottles at 5000rpm for 10 min at 4oC.
Make sure the seals on the bottle are properly seated and
the bottles are balanced within 0.1g.
Carefully decant the supernatant down the drain.
*You may replace the cap and store the pellet at -20oC.
*Before going on with the prep, be sure the ultracentrifuge and rotor are available
and sign up to use them.
You'll do one 30 min spin and one for 36 hours!

Prepare lysozyme on put on ice.
You'll need 0.5ml per culture.
Use plastic tube, and don't vortex.
For 1ml: 0.75ml water
0.25ml 1M Tris-Cl, pH 7-8
10mg lysozyme (stored in desiccator at -20oC)

If the pellet isn't soft , vortex it for a long time until it is.

Add to pellet 3ml 25% sucrose, 50mM Tris-Cl. Mix
Add 0.5ml lysozyme. Mix gently. Leave on ice for 5 min.

Add 1ml 0.5M EDTA, leave on ice for 5 min.

Add 4.5ml Triton juice. Mix gently.
[Triton juice: 4ml 10% Triton-X100, 20ml 0.5M EDTA, 20ml 1M Tris-Cl, 320ml water]
Pour the mess carefully into clean centrifuge tubes.
Put on ice and watch for at least 10 min for the solution to become viscous.
Take the cap off and see the snotty DNA.
Balance the tubes to within 0.01g.

Spin the tubes at 30,000rpm for 30 min at 4oC.
Make sure the rotor is clean and dry, and that the O-ring is seated properly.

Decant the supernatant into a 15ml disposable tube. Don't let pellet fall.
Estimate the volume by the gradations on the tube to the nearest 0.1ml.
Add 0.95g CsCl per ml. Invert gently until completely dissolved.

Into disposable ultracentrifuge tubes, pipet ethidium bromide. Wear gloves.
Approximately 200ul of 10mg/ml ethidium bromide per 10ml of CsCl/DNA solution.

Pipet the CsCl/DNA solution into the tubes. Use a pasteur pipet as a funnel.
Bring the volume up to the neck with a separate solution of CsCl.
This is made by adding 0.95g CsCl per ml to 25% sucrose, 50mM Tris.

Balance the tubes to within 0.01g. Seal the tubes.

Put the tubes in a clean,dry rotor.

If necessary, cover the tubes with metal caps that weigh the same.
Make sure the O-ring is seated properly.

Spin 50,000rpm at 15oC for about 36 hours.

Break down to 1000-800rpm, then let brake off.

Remove tubes carefully. Wear gloves.
Gently open the tubes at the top.
Insert a 21-20G needle in 3ml syringe just below lower band.
Pull lower band, remove needle carefully, put solution into polyallomer 5ml tube.
Empty tube into bleach. Discard needle in sharps bucket.

Extract solution at least twice with isopropanol over salt-saturated water.
Dialyze or precipitate the DNA. To precipitate, double the volume with TE;
add 2 volumes ethanol; ice, spin.