[Triton-Lysozyme Lysis-PEG precipitation - Centrifugation in CsCl/ EtBr Gradients]

• Sucrose buffer
  75 gm Sucrose
  25 ml 1 M TRIS pH 7.5
  
  
• 50 ml 0.5M EDTA pH 7.5
  Adjust to 500 ml in H₂O
  
  
• Triton X buffer
  1 ml Triton X-100
  50 ml 1 M TRIS pH 8.0
  100 ml 0.5 M EDTA pH 7.5
  Adjust to 1 liter
  
  
• PEG solution
  150 gm PEG
  43.84 gm NaCl
  Adjust to 500 ml
  
  
• "Red juice"
  221.6 gm CsCl
  210.0 ml TE (10 mM TRIS, 1 mM EDTA , pH 7.6)
  0.8 ml EtBr (10 mg/ml)

1. Inoculate bacteria in 500 ml of L broth containing appropriate antibiotic and incubate overnight at 37°C with shaking.
   [If low-copy number plasmids are prepared, plasmid amplification using chloramphenicol should be performed as procedure described in Molecular Cloning: A Laboratory Manual by Maniatis et al.]
   
2. Harvest bacterial cells by centrifugation at 5000 RPM in Sorvall centrifuge for 15 minutes at 4°C, then pour off supernatant and put bottle up side down on paper towel to remove media.
   
3. Resuspend bacterial pellet in 15 ml of Sucrose buffer and transfer into 400 ml Sorvall tube.
   
4. Add 5 ml of 20 mg/ml freshly prepared lysozyme in sucrose buffer, mix and incubate for 30 minutes on ice.  Invert periodically.
   
5. Add 12 ml of Triton X 100 buffer and incubate at 37°C 30 minutes to lyse bacterial cell wall.
   
6. Isolate plasmid from cell debris by centrifugation at 15,000 RPM for at least one hour.
   
7. Pour off supernatant into 50 ml Falcon tube and add half volume of PEG solution then mix and incubate on ice for 30 minutes, or overnight, to precipitate plasmid DNA.
   
8. Centrifuge at 2500 RPM for 15 minuts and discard supernatant (use cotton swab to remove residual PEG solution).
   
9. Resuspend pellet in "Red juice" containg CsCl and EtBr. (volume 6- 10 ml)
10. Transfer into Quick-Seal centrifuge tubes (Beckman) for ultracentrifugation, heat seal after filling the tubes.
    
11. Spin for at least 18 hours at 55,000 RPM, 20°C using Ti 70.1 rotor.
    
12. Collect the band of circular plasmid DNA (the lower band) under long wavelength UV lamp using needle and syringe.
    
13. Remove EtBr by extracting several times with an equal volume of isopropanol saturated with1X TE and CsCl (or extract with Butanol) until the red color is gone in the DNA phase (bottom).
    
14. Dialyse DNA in TE to remove CsCl. Four changes of TE over 1 - 2 days. in 2 or 4 liters.