8. Centrifuge and rotor similar to Thermo Sorvall Legend XTR
with Thermo Scientific Swing-out rotor and corresponding
adapters and tubes.
9. Centrifuge and rotors similar to Thermo Sorvall Lynx 4000
with either F21-8 Â 50y or F14-6 Â 250y rotor and
corresponding tubes or bottles.
10. Mixer Mill MM 400 (Retsch), along with grinding jars and
grinding balls.
11. Spatulas, tongs, and tweezers.
2.4 Purification
of Crude Ribosomes
1. 500 mL low-salt HMA buffer: (see Subheading 2.3, item 6).
Filter-sterilize the solution and store at 4
C.
2. 500 mL high-salt HMA buffer: 20 mM HEPES-KOH pH 7.5,
600 mM NH 4 Cl, 10 mM MgCl 2 , 5 mM BME. Add DEPCtreated water (see Subheading 2.3, item 1) to the final volume
of 500 mL. Filter-sterilize the solution and store at 4
C.
3. DNase (TURBO, 2 U/mL).
4. Thermo Sorvall Lynx 4000 Centrifuge along with
F21-8 Â 50y and 50 mL tubes.
5. Wheaton Potter-Elvehjem-type 30 mL homogenizer (Sigma
Aldrich).
6. Ultracentrifuge similar to Beckman Coulter Optima L-90K.
7. Ultracentrifuge rotor similar to TY70Ti (Beckman Coulter)
and corresponding 23.6 mL polycarbonate tubes with cap
assemblies.
8. Benchtop refrigerated centrifuge similar to Eppendorf 5417R.
9. UV spectrophotometer.
2.5 Preparation
of Continuous 10–40%
Sucrose Gradient
1. 40 mL polypropylene ultracentrifuge tubes.
2. 500 mL 10% sucrose in low-salt HMA buffer: 20 mM HEPESKOH pH 7.5, 30 mM NH 4 Cl, 10 mM MgCl 2 , 5 mM BME,
50 g of sucrose. Add DEPC-treated water to the final volume
of 500 mL. Filter-sterilize the solution and store at 4
C.
3. 500 mL 40% sucrose in low-salt HMA buffer: 20 mM HEPES-K
pH 7.5, 30 mM NH 4 Cl, 10 mM MgCl 2 , 5 mM BME, 200 g of
sucrose. Add DEPC-treated water to the final volume of
500 mL. Filter-sterilize the solution and store at 4
C.
4. Gradient maker (Biocomp Gradient Station).
2.6 Sucrose Gradient
Ultracentrifugation
and Fractionation
1. 500 mL low-salt HMA buffer (see Subheading 2.3, item 6).
Filter-sterilize the solution.
2. 40 mL polypropylene tubes (Beckman Coulter) filled with
38.5 mL of 10–40% sucrose gradient (see Subheading 3.5).
3. Ultracentrifuge similar to Beckman Coulter Optima L-90K.
Purification of Three Types of Ribosomes from Mycobacteria
155
with Thermo Scientific Swing-out rotor and corresponding
adapters and tubes.
9. Centrifuge and rotors similar to Thermo Sorvall Lynx 4000
with either F21-8 Â 50y or F14-6 Â 250y rotor and
corresponding tubes or bottles.
10. Mixer Mill MM 400 (Retsch), along with grinding jars and
grinding balls.
11. Spatulas, tongs, and tweezers.
2.4 Purification
of Crude Ribosomes
1. 500 mL low-salt HMA buffer: (see Subheading 2.3, item 6).
Filter-sterilize the solution and store at 4
C.
2. 500 mL high-salt HMA buffer: 20 mM HEPES-KOH pH 7.5,
600 mM NH 4 Cl, 10 mM MgCl 2 , 5 mM BME. Add DEPCtreated water (see Subheading 2.3, item 1) to the final volume
of 500 mL. Filter-sterilize the solution and store at 4
C.
3. DNase (TURBO, 2 U/mL).
4. Thermo Sorvall Lynx 4000 Centrifuge along with
F21-8 Â 50y and 50 mL tubes.
5. Wheaton Potter-Elvehjem-type 30 mL homogenizer (Sigma
Aldrich).
6. Ultracentrifuge similar to Beckman Coulter Optima L-90K.
7. Ultracentrifuge rotor similar to TY70Ti (Beckman Coulter)
and corresponding 23.6 mL polycarbonate tubes with cap
assemblies.
8. Benchtop refrigerated centrifuge similar to Eppendorf 5417R.
9. UV spectrophotometer.
2.5 Preparation
of Continuous 10–40%
Sucrose Gradient
1. 40 mL polypropylene ultracentrifuge tubes.
2. 500 mL 10% sucrose in low-salt HMA buffer: 20 mM HEPESKOH pH 7.5, 30 mM NH 4 Cl, 10 mM MgCl 2 , 5 mM BME,
50 g of sucrose. Add DEPC-treated water to the final volume
of 500 mL. Filter-sterilize the solution and store at 4
C.
3. 500 mL 40% sucrose in low-salt HMA buffer: 20 mM HEPES-K
pH 7.5, 30 mM NH 4 Cl, 10 mM MgCl 2 , 5 mM BME, 200 g of
sucrose. Add DEPC-treated water to the final volume of
500 mL. Filter-sterilize the solution and store at 4
C.
4. Gradient maker (Biocomp Gradient Station).
2.6 Sucrose Gradient
Ultracentrifugation
and Fractionation
1. 500 mL low-salt HMA buffer (see Subheading 2.3, item 6).
Filter-sterilize the solution.
2. 40 mL polypropylene tubes (Beckman Coulter) filled with
38.5 mL of 10–40% sucrose gradient (see Subheading 3.5).
3. Ultracentrifuge similar to Beckman Coulter Optima L-90K.
Purification of Three Types of Ribosomes from Mycobacteria
155