| Contributor: |
The Laboratory of Giovanna Ferro-Luzzi Ames at the University of California, Berkeley
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1. Grow a 50 ml bacterial culture in LB overnight.
2. Centrifuge to pellet cells (approximately 1,500 X g) and discard the supernatant.
3. Resuspend the pelleted cells in 500 μl cold Tris Buffer 1.
4. Transfer cell solution to a thick walled Pyrex tube.
5. Add 100 μl of Tris Buffer 2
6. Incubate on ice for 10 min.
7. Add 200 μl 250 mM EDTA
8. Incubate on ice for 10 min.
9. Add 10 μl of RNase A and 10 μl of RNase T solution.
10. Incubate on ice for 10 min.
11. Add 800 μl of Triton Solution.
12. Incubate on ice for 10 min.
13. Centrifuge at 18,000 X g for 45 min at 4°C.
14. Remove the clear lysate using a Pasteur pipette.
15. Store the lysate at -20°C or -80°C for long term storage.
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| RNase A |
| 1 mg/ml boiled RNase A
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| 250 mM EDTA |
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| Tris Buffer 2 |
| 250 mM Tris, pH 8.0
Prepare just before use
10 mg/ml Lysozyme
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| Tris Buffer 1 |
| 25% (w/v) Sucrose
50 mM Tris, pH 8.0
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| Luria Broth (LB) |
| 5g/liter Yeast Extract
10g/liter Tryptone
1ml/liter 1 M NaOH
5g/liter NaCl
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| 1 M NaOH |
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| Triton Solution |
| 50 mM EDTA
50 mM Tris, pH 8.0
0.1 % (w/v) Triton X 100
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| RNase T |
| 1 mg/ml RNase T1
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Sucrose
Triton X-100
Sodium Hydroxide
EDTA
Lysozyme
Yeast Extract
Tris
Tryptone
Sodium Chloride
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No hints are associated with this bioProtocol
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