Purification of 70S Ribosomes from Bacillus subtilis

Materials and Reagents

1. Bacillus subtilis 168
   
2. NaCl
   
3. BD Bacto^TM tryptone (Difco, catalog number: 211705 )
   
4. BD Bacto^TM yeast extract (Difco, catalog number: 212750 )
   
5. Agar (Nissui, catalog number: 05835 )
   
6. Tris (Wako Pure Chemical Industries, catalog number: 512-97505 )
   
7. Magnesium acetate (Wako Pure Chemical Industries, catalog number: 139-15335 )
   
8. Ammonium acetate (Wako Pure Chemical Industries, catalog number: 019-02835 )
   
9. Dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 042-29222 )
   
10. Phenylmethylsulphonyl fluoride (PMSF)
    
11. Sucrose (Sigma-Aldrich, catalog number: S0389-55G )
    
12. Diethylpyrocarbonate (DEPC) treated water
    Note: DEPC was added to distilled water at a final concentration of 1%, and then it stored at room temperature for over two hours. The DEPC-treated distilled water was autoclaved twice (121 °C, 20 min) and stored at room temperature.
    
13. LB medium (see Recipes)
    
14. Buffer I (see Recipes)
    
15. 10-40% sucrose solutions (see Recipes)
    

Equipment

1. 2 L flasks with cotton plugs
   
2. Innova 4080 bench top incubator shaker (New Brunswick scientific)
   
3. French pressure cell press [Aminco International (USA), model: FA-078 ]
   
4. Mini cell [Aminco International (USA), model: FA-003 ]
   
5. Gradient fractionator (BIOCOMP, catalog number: 152-001 )
   
6. Gradient master [Aminco International (USA), catalog number: 107-201M ]
   
7. Micro collector (ATTO Technology, catalog number: AC-5700P )
   
8. Nano drop 2000 (Thermo Fisher Scientific)
   
9. High-speed refrigerated centrifuge (e.g. Hitachi, model: himac CR22GII )
   
10. Angle rotor R10A3 (fixed angle, max: 10,000 rpm,18,800 x g, volume: 500 ml x 6) (Hitachi)
    
11. Centrifuge MX-300 (TOMY)
    
12. TMA-300 rotor (fixed angle, max: 15,000 rpm, 20,380 x g, volume: 2 ml x 24) (TOMY)
    
13. TMA-27 rotor (fixed angle, max: 15,000 rpm, 21,130 x g, volume: 15 ml x 4, 50 ml x 4) (TOMY)
    
14. Centrifuge tube (Nalgene, catalog number: 3148-0050 )
    
15. Ultra-centrifuge (e.g. Hitachi, model: CP-60E )
    
16. P55ST2 rotor (swinging bucket rotor, max: 55,000 rpm, 366,000 x g, volume: 5 ml x 6) (Hitachi)
    
17. P28S rotor (swinging bucket rotor, max: 28,000 rpm, 141,000 x g, volume: 40 ml x 6) (Hitachi)
    
18. P40ST rotor (swinging bucket rotor, max: 40,000 rpm, 284,000 x g, volume: 13 ml x 6) (Hitachi)
    
19. Open-top polyclear centrifuge tube for P40ST rotor (Seton Identification Products, part number: 7031 )
    
20. Open-top polyclear centrifuge tube for P28S rotor (Seton Identification Products, part number: 7052 )
    
21. Centrifuge ware for P55ST2 rotor (Hitachi, part number: 332245A )
    Note: These rotors can substitute for other commercial rotors which can be used by the same centrifugal force as described in procedures.
    

Procedure