1) Grow 500ml cells at 37°C until OD600 rm = 0.6. Add 1.65ml of 50mg/ml chloramphenicol in 100% ethanol to give a final concentration of 167µg/ml. Incubate overnight (12-15 hours) at 37°C.
2) Harvest the cells at 5K for 10 min. Drain well.
3) Suspend the pellets in 18ml of Solution I. Add 2 ml of Solution I containing 20mg/ml lysozyme (fresh). Incubate at room temperature for 10 min. (Note: if the cell culture was not amplified with chloramphenicol, use 40mg/ml lysozyme.)
4) Add 40ml of Solution II. Mix gently by swirling. Place in an ice water bath for 5 min.
5) Add 20ml of pre-chilled Solution III. Mix the slurry by swirling. Incubate on ice for 15-20 min.
6) Centrifuge at 10K in a GSA rotor for 10 min. Carefully decant the supernatant from the pelleted cell debris. Filter through a Kimwipe in a funnel to remove any debris.
7) Add 0.6 volume of isopropanol (43.5ml). Mix by swirling. Let stand on ice for 20 min.
8) Centrifuge for 15 min. at 10K in a GSA. Discard supernatant. Let the pellets dry briefly (until the isopropanol has evaporated).
9) Dissolve the pellet in 9.3ml TE. Add 10g CsCl and 0.7ml EtBr (10mg/ml).
10) Centrifuge at 38-40K, 20°C for 48 hr in 70.1Ti rotor.
11) Remove the bands (visualized by either long UV light or visible light) containing plasmid DNA. Extract repeatedly (minimum 6X) with isopropanol (saturated with CsCl and H2O) until the orange-red color of ethidium bromide is no longer visible.
12) Remove the CsCl by exhaustive dialysis overnight against TE at 4°C.
13) Add 1/10 volume of 3M NaOAc and 3 volumes of 100% ethanol. Chill at -70°C for 10 min and centrifuge at 17,000 x g for 15 min. Decant supernatant. Drain well.
Solution I (50mM glucose, 25mM Tris pH8 10mM EDTA pH8)
9.01g Glucose
25ml 1M Tris pH8.0
40ml 250mMEDTA pH8.0
DD H2O to 1 liter
Autoclave for 20 min.
Solution II
0.2M NaOH
1% SDS
Make fresh.
Solution III
600ml 5M Potassium Acetate (pH4.8) (= 300g/600ml)
115ml Glacial Acetic Acid
285ml DD H2O
Autoclave for 20 min.