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The Laboratory of Dan Herschlag at Stanford University
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| This protocol describes the preparation of ribosomes or different species of ribosomal associations from bacterial cells. It is mainly intended for preparative purposes; it should not be used to assess the relative abundance of various ribosomal species in the cell because the freeze/thaw lysis can give artifactual results. |
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1. Streak out the E. coli strain of interest to single colonies.
2. Inoculate 5 ml of LB Medium with a bacterial colony.
3. Grow the culture overnight at 37°C with shaking.
4. Inoculate 100 ml of LB Medium with 2 ml from the overnight culture.
5. Grow with shaking at 37°C until the optical density at 600 nm (OD600) is between 0.4 to 0.5.
6. Add Chloramphenicol to a final concentration of 0.1 mg/ml three min prior to harvesting the cells (see Hint #1).
7. Pour the cell culture over an equal mass of ice in centrifuge tubes.
8. Centrifuge at 8,000 rpm in a JA-14 or equivalent rotor (10,000 X g) for 10 min at 4°C to harvest the cells.
9. Decant the supernatant. Use a Pasteur pipette to remove the last bit of supernatant.
10. Resuspend the pellet in 0.5 ml of ice-cold Buffer A. Transfer the cell suspension to a microcentrifuge tube.
11. Add 50 μl of Lysozyme Solution. Mix by pipetting up and down 3 times.
12. Freeze the bacterial suspension for 5 min in a Dry Ice-Ethanol bath or in Liquid Nitrogen. The sample can be stored at -80°C if desired.
13. Thaw in an ice water bath. For faster thawing, use a cool water bath instead.
14. Repeat the freeze-thaw cycle (Steps #12 through #13).
15. Add 15 μl of 10% Deoxycholate to complete the cell lysis.
16. Centrifuge the lysate at top speed in a microcentrifuge for 10 min at 4°C to pellet the cell wall debris and DNA (see Hint #2).
17. Collect the supernatant. It can be stored at -80°C if desired. Keep on ice until ready to load on the Sucrose gradient to prevent degradation by proteases and RNases.
18. Pour a 10 ml 10% to 40% Sucrose gradient in an SW41 Ti polyallomer tube. This can be accomplished by pouring a linear 10% to 40% gradient using a gradient maker with one chamber filled with 5 ml of 10% Sucrose Gradient Solution and the other with 5 ml of 40% Sucrose Gradient Solution. Alternatively, set up a step gradient consisting of 2.5 ml layers of 10%, 20%, 30%, and 40% Sucrose Gradient Solutions and allow the gradient to form overnight in a cold room.
19. Determine the absorbance at 260 nm (Abs260) of a 1:500 dilution of the supernatant to determine the RNA content. Assume that 1 Abs260 = 40 μg/ml RNA.
20. Gently load between 0.5 to 1.5 mg RNA on the gradient on ice. Balance the tube with cold Buffer A.
21. Centrifuge the gradient in an SW41 Ti rotor at 35,000 rpm (151,000 X g) with the brake on for 2.5 hr at 4°C.
22. Collect 0.5 ml fractions from the gradient.
23. Determine the Abs260 of the fractions to determine which fractions contain the highest levels of RNA - these are the ones that contain ribosomal RNA. Store the peak RNA fractions at -80°C.
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| Buffer A |
| 100 mM Ammonium Chloride (NH4Cl)
10 mM MgCl2
20 mM Tris-Cl, pH 7.5
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| Chloramphenicol |
| Prepare in 100% Ethanol
10 mg/ml Chloramphenicol
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| LB Medium |
| 5g/liter Yeast Extract
1 ml/liter 1 M NaOH (CAUTION! see Hint #3)
Autoclave
10g/liter Tryptone
5g/liter NaCl
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| 1 M NaOH |
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| 40% Sucrose Gradient Solution |
| Prepare in Buffer A With 2-Mercapoethanol
40% (w/v) Sucrose
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| 10% Sucrose Gradient Solution |
| Prepare in Buffer A With 2-Mercapoethanol
10% (w/v) Sucrose
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| 10% Deoxycholate |
| 10% (w/v) Sodium Deoxycholate
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| Lysozyme Solution |
| 10 mg/ml Lysozyme
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| Buffer A With 2-Mercapoethanol |
| 100 mM Ammonium Chloride (NH4Cl)
Filter sterilize to remove particulates
10 mM MgCl2
20 mM Tris-Cl, pH 7.5
2 mM 2-Mercaptoethanol
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Dry Ice
Ammonium Chloride
Lysozyme
Yeast Extract
Tryptone
Deoxycholate
Sucrose
Tris
Ethanol
Magnesium Chloride
2-Mercaptoethanol
Sodium Chloride
Chloramphenicol
Sodium Hydroxide
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1. This is to stop translational elongation and prevent ribosomes running off the mRNA. If you want only free ribosomes then skip this step. The next step (cooling) will inhibit initiation of translation but not elongation.
2. It is also possible to centrifuge the lysate in a tabletop centrifuge for 20 to 30 min. However, the supernatant should be centrifuged a second time to clarify it. It is important that particulate matter and DNA are removed from the lysate before it is loaded on the sucrose gradient to prevent smearing of the gradient.
3. CAUTION! This substance is a biohazard. Please consult with this agent's MSDS for proper handling instructions.
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1. Dammel CS and Noller HF. Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA. Genes Dev. 1995; 9:626-37.
4. Ron EZ, Kohler RE, Davis BD Polysomes extracted from Escherichia coli by freeze-thaw-lysozyme lysis. Science. 1966; 153:1119-20.
3. Powers T and Noller HF. Dominant lethal mutations in a conserved loop in 16S rRNA. Proc Natl Acad Sci U S A. 1990; 87:1042-6.
2. Dammel CS and Noller HF. A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly. Genes Dev. 1993; 7:660-70.
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