HIGH COPY NUMBER PLASMID PREP FOR TL 100

1. Grow 50 mls of bacterials cells to late logarithmic phase.
2. Centrifuge cells at 5000g for 5 minutes.
3. Resuspend cells in 1.5 mls lysozyme solution.
4. Keep on ice for 5 minutes.
5. Add 3.5 ml of 0.2N NaOH, 1% SDS
6. vortex, ice for 30 minutes.
7. Add 3.5 ml of 2M NaOAc (pH 4.8).
8. Centrifuge 40,000g for 30 minutes.
9. Decant supernatant; add 20 mls cold 95% ethanol.
10. Precipitate at -20 C
11. Centrifuge 5,000g for 15 minutes.
12. Resuspend in 1.5 mls distilled water.
13. Adjust volume to 1.7 mls, add 2.0 g CsCl, 80 ul EtBr
14. Centrifuge 100,000 rpm for 6 hrs minimum (20 C)

NOTES:

1. lysozyme sol'n is: 50 mM glucose, 10 mM EDTA, 25 mM Tris-Cl (8.0)

   to make 10 mls:		7.30 mls     x     dd H2O
                           0.20 mls     x     0.5 M EDTA
                           0.25 mls     x     1.0 M  Tris-Cl (8.0)
   

   then, to 6 mls of above, add 1 ml of 120 mg/ml lysozyme made fresh.
2. 0.2N NaOH, 1% SDS must be made fresh each time.
3. 2.0M NaOAc must be at pH=4.8 (critical)
4. add 80 ul of EtBr (10 mg/ml) directly to TL 100 quick-seal tube, then add the DNA/CsCl to top of tube.