Lysis-by-Alkali Plasmid Prep
v1.3, 2/22/94

1. Add 1ml of a fresh overnight culture to 500ml of LB broth+antibiotic.
Grow @ 37°C for ~24hrs.
2. Split the 500ml culture into two 250ml Sepcor centrifuge bottles.
Harvest cells by centrifugation @ 5000rpm for 15min @ 4°C in 250ml bottles in the GS3 rotor. Pour off supernatant.
Invert bottle to allow supernatant to drain away.
3. Resuspend in 50ml ice-cold STE.
0.1M NaCl
10mM Tris·Cl(pH 8.0)
1mM EDTA(pH 8.0)
4. Centrifuge 5000rpm @ 4°C for 15min.
5. Resuspend the pellet in 9ml of Solution I.
50mM Glucose
25mM Tris·Cl(pH 8.0)
10mM EDTA(pH 8.0)
Combine the two resuspensions in one of the bottles.
6. Add 2ml of freshly prepared lysozyme.

10mg/ml lysozyme
10mM Tris·Cl(ph 8.0)
7. Add 40ml of freshly prepared Solution II.
0.2N NaOH(from 10N stock)
1% SDS
Close the bottle add mix thoroughly by inverting the bottle several times.
Store the bottle at room temp for 10 min.
8. Add 20ml ice-cold Solution III.
5M Potassium Acetate 60ml Glacial acetic acid 11.5ml H20 28.5ml
Close the bottle and mix by shaking. Store on ice for 10 min. A flocculent white precipitate should form.
9. Centrifuge the lysate @ 5000rpm for 20 min @ 4°C.
Use a 25ml pipet to remove as much of the supernatant as possible while avoiding the floating debris. Note the amount of the recovered volume.
Add 0.6 volume of isopropanol, mix, and store at room temp for 10 min.
10. Recover the nucleic acids by centrifugation @ 5000rpm for 15 min @ room temp. (Salt may precipitate @ 4°C)
11. Decant supernatant carefully and invert the bottle to allow any drops to drain away. Rinse the pellet and the walls of the bottle with 70% EtOH @ room temp. Drain off EtOH and use a vacuum line to remove any drops. Let stand inverted to allow EtOH to evaporate.

12. Resuspend pellet of nucleic acids in 3ml TE(pH 8.0).
13. Purify the plasmid DNA by equilibrium centrifugation in CsCl gradients.
Add 1g of solid CsCl for each ml of DNA solution. Warm to 30°C to facilitate dissolution of the salt.
14. Add 0.8ml of ethidium bromide(10mg/ml in water) for every 10ml of DNA/CsCl solution. Immediately mix. Store on ice 10 min.
15. Centrifuge @ 8000rpm for 5min at room temp.
16. Transfer the supernatant to a Beckman Quick-Seal ultracentrifuge tube using a syringe. Bring the tubes to volume with CsCl sol'n. The density of the sol'n in the tube should be ~1.55g/ml.
Balance the tubes to within ±0.02gm. Seal the tubes.
Check the tubes for leaks.
17. Centrifuge in a Vti-80 rotor or equivalent @ 55000rpm for 14 hrs.
18. After centrifugation, two bands should be visible. Remove the lower band with a needle and syringe using UV to illuminate the bands if necessary.
Transfer to a small test tube.
19. Add equal volume of 1-butanol or isoamyl alcohol and mix well. Remove upper layer to a waste collection tube.
Repeat until neither layer shows any pink color.
Resin columns can be used as an alternate method to organic solvents in this step.
20. Add 3 volumes of water and 2x total(water+DNA) absolute EtOH. Mix and store 10 min @ 4°C. Spin 15 min @ 10000xg @ 4°C.
21. Resuspend in 1ml TE(pH 8.0). Measure OD280 and OD260.